Hot topics: Cannabinoid receptor stuctures

Two new reports cover further aspects of cannabinoid receptor binding as defined using cryo-EM. These expand on previous reports of the structure of the human CB1 cannabinoid receptor with the antagonists taranabant and  AM6538 bound, and with the agonists MDMB-FUBINACA, AM11542 and AM841 bound. Previously reported also is the structure of the CB2 cannabinoid receptor with the antagonist AM10257 bound.

In the first report (1), a non-selective, high potency agonist WIN55212-2 was bound to a complex of the CB2 cannabinoid receptor with Gi to “complete the set” of agonist- and antagonist-bound versions of CB1 and CB2 receptors. The authors suggest marked similarities in the binding configuration of the agonist WIN55212-2 and the antagonist AM10257. Based on these structures, two novel ligands were generated; one predicted to be an agonist and the other an antagonist. The functional data supported these predictions, although both novel compounds were markedly less potent than their respective positive controls.

In the second report (2), agonist-bound versions of both CB1 and CB2 cannabinoid receptors complexed with Gi were also described to ‘complete and expand the set’. AM12033 is a novel nitrilodimethylheptyl analogue of THC with high affinity at both receptors. Structures using this compound were compared to CB1 receptors bound to the THC-like AM841. The authors suggest similar modes of interaction for agonists at the two receptors, supported by numerous examples of non-selective high potency agonists.

Modelling of the second intracellular loop in both reports suggested a structural basis for the CB2 receptor to couple poorly to Gs. Indeed, a single amino acid variation (L222 in CB1 and P139 in CB2 receptors) was highlighted in both studies and evidenced in the previous literature (https://www.ncbi.nlm.nih.gov/pubmed/23667597).

Comments by Prof. Steve Alexander (@mqzspa), University of Nottingham, UK

(1) Xing C. et al. Cryo-EM Structure of the Human Cannabinoid Receptor CB2-G i Signaling Complex. Cell (2020) doi:10.1016/j.cell.2020.01.007 [PMID:32004460]

(2) Hua T. et al. Activation and Signaling Mechanism Revealed by Cannabinoid Receptor-Gi Complex Structures. Cell  (2020) doi:10.1016/j.cell.2020.01.008 [PMID:32004463]

 

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Hot Topics: A brief update on coronaviruses

The new respiratory coronavirus from Wuhan in China is causing intense research activity in the world. Viruses are evidently associated with respiratory infections ranging in severity from the common cold to pneumonia and death. More than 200 viral types have been associated with the common cold, of which 50% of infections are rhinovirus, but also respiratory syncitial virus, influenza – and coronaviruses, particularly human coronavirus-229E (HCoV-229E), which is ‘relatively benign’. Monocytes are relatively resistant to HcoV-229E infection, but rapidly invades, replicates in, and kills dendritic cells within a few hours of infection (Mesel-Lemoine et al., 2012) [1]. Dendritic cells are the sentinel cells in the respiratory tract, and plasmacytoid dendritic cells are a crucial antiviral defence via interferon production. Thus, these viruses can impair control of viral dissemination and the formation of long-lasting immune memory. The cellular receptor for HcoV-229E is CD-13 (aminopeptidase-N).
The cellular receptors for coronaviruses are critical for cell entry. Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), and HCoV-NL63, bind to angiotensin converting enzyme2, ACE2, on ciliated cells in the respiratory tract, the crystal structure is known (Song et al., 2018) [2]. The SARS-CoV spike (S) glycoprotein (with S1 and S2 sub-units) on the outer envelope binds to ACE2, fusing viral and cellular membranes, triggering conformational transformations. Cleavage of the S1/S2 subunits by proteases is critical. Indeed, Simmons et al (2005) [3] showed that SARS-CoV infection involved receptor binding, conformational changes receptor binding and subsequent cathepsin L (and type II transmembrane serine proteases) proteolysis within endosomes – with inhibitors of cathepsin L preventing viral entry. SARS-CoV may lead to markedly elevated cytokine levels, leading to tissue damage, pneumonitis, and acute respiratory distress syndrome (ARDS). The spike proteins in SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) are crucial for host specificity and jumping between species, e.g. from bats to humans (Lu et al., 2015) [4], and also the recent cross-over to swine acute diarrhoea syndrome coronavirus (SADS-CoV) to pigs (Zhou et al., 2018) [5]. There is intense interest in the complicated ways whereby coronaviruses engage with their target receptors, are activated and replicate. The rapid evolution of coronaviruses is critical as humans have little or no pre-existing immunity SARS- or MERS-CoV.

ACE2 has been identified as a potential receptor for the novel coronavirus that was identified as the cause of the respiratory disease outbreak in Wuhan in late 2019 (referred to as 2019 – nCoV by the WHO, or Wuhan coronavirus) [6,7 & 9]. 2019-nCoV is a betacoronavirus, and, in common with the SARS-CoV [8], the viral spike protein is postulated to engage ACE2 for viral entry [7]. The data presented by Letko and Munzter [7] is preliminary and has not been peer-reviewed, but a rapid pace of research is needed in the present circumstances. For example, in another recent, preliminary report, ACE2 receptor expression is highly expressed in type II alveolar cells (AT2) with much individual variation and the authors reported that the cells also expressed many genes involved in viral reproduction and transmission [10]. While these findings are preliminary, the rapid publications in bioRxiv and similar archives are crucial in what is now officially a global health emergency. This report is a hot topic in the IUPHAR/BPS Guide to PHARMACOLOGY (https://www.guidetopharmacology.org), and further underlines the importance of immunopharmacology and the IUPHAR Guide to IMMUNOPHARMACOLOGY (https://www.guidetoimmunopharmacology.org/immuno/index.jsp).

Comments by Prof. Michael Spedding, Spedding Reseach Solutions; Prof. Steve Alexander (@mqzspa), University of Nottingham; Dr. Elena Faccenda, University of Edinburgh; and Prof. Francesca Levi-Schaffer, The Hebrew University of Jerusalem.

Note from bioRxiv: bioRxiv is receiving many new papers on coronavirus 2019-nCoV.   A reminder: these are preliminary reports that have not been peer-reviewed. They should not be regarded as conclusive, guide clinical practice/health-related behavior, or be reported in news media as established information.

 

  1. Mesel-Lemoine M, Millet J, Vidalain PO, Law H, Vabret A, Lorin V, Escriou N, Albert ML, Nal B, Tangy F. A human coronavirus responsible for the common cold massively kills dendritic cells but not monocytes. J Virol. 2012 Jul;86(14):7577-87. doi: 10.1128/JVI.00269-12. Epub 2012 May 2. PubMed PMID: 22553325; PubMed Central PMCID: PMC3416289.
  2. Song W, Gui M, Wang X, Xiang Y. Cryo-EM structure of the SARS coronavirus spike glycoprotein in complex with its host cell receptor ACE2. PLoS Pathog. 2018 Aug 13;14(8):e1007236. doi: 10.1371/journal.ppat.1007236. eCollection 2018 Aug. PubMed PMID: 30102747; PubMed Central PMCID: PMC6107290.
  3. Simmons G, Gosalia DN, Rennekamp AJ, Reeves JD, Diamond SL, Bates P. Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entry. Proc Natl Acad Sci U S A. 2005 Aug 16;102(33):11876-81. Epub 2005 Aug 4. PubMed PMID: 16081529; PubMed Central PMCID: PMC1188015.
  4. Lu R, Wang Y, Wang W, Nie K, Zhao Y, Su J, Deng Y, Zhou W, Li Y, Wang H, Wang W, Ke C, Ma X, Wu G, Tan W. Complete Genome Sequence of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) from the First Imported MERS-CoV Case in China. Genome Announc. 2015 Aug 13;3(4). pii: e00818-15. doi: 10.1128/genomeA.00818-15. PubMed PMID: 26272560; PubMed Central PMCID: PMC4536671.
  5. Zhou L, Sun Y, Lan T, Wu R, Chen J, Wu Z, Xie Q, Zhang X, Ma J. Retrospective detection and phylogenetic analysis of swine acute diarrhoea syndrome coronavirus in pigs in southern China. Transbound Emerg Dis. 2019 Mar;66(2):687-695. doi: 10.1111/tbed.13008. Epub 2019 Jan 9. PubMed PMID: 30171801.
  6. Gralinski LE, Menachery VD. Return of the Coronavirus: 2019-nCoV. Viruses. 2020 Jan 24;12(2). pii: E135. doi: 10.3390/v12020135. PubMed PMID: 31991541.
  7. Letko M, Munster V. Functional assessment of cell entry and receptor usage for lineage B β coronaviruses, including 2019-nCoV. bioRxiv 2020.01.22.915660; doi: 10.1101/2020.01.22.915660
  8. Li F, Li W, Farzan M, Harrison SC. Structure of SARS coronavirus spike receptor-binding domain complexed with receptor. Science. 2005 Sep 16;309(5742):1864-8. PubMed PMID: 16166518.
  9. Peng Z, Shi Z-L. (2020) Discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin. bioRxiv 2020.01.22.914952; doi: 10.1101/2020.01.22.914952
  10. Yu Zhao, Zixian Zhao, Yujia Wang, Yueqing Zhou, Yu Ma, Wei Zuo.Single-cell RNA expression profiling of ACE2, the putative receptor of Wuhan 2019-nCov bioRxiv 2020.01.26.919985 doi: 10.1101/2020.01.26.919985
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Hot Topics: Cryo-EM structure of a selective T-type calcium channel blocker bound to the Cav3.1 voltage-gated calcium channel

Nieng Yan’s group has now published the cryo-EM structure of the selective T-type Ca2+ channel blocker Z944 bound to the pore-forming α1-subunit of Cav3.1 T-type Ca2+ channels (1). This nicely adds to recent publications reporting of the high-resolution structures of L-type Ca2+ channel blockers (nifedipine, nimodipine, amlodipine, verapamil, diltiazem) bound to the rabbit Cav1.1 skeletal muscle Ca2+ channel (2) and to the model Ca2+ channel CavAb (Ca2+-selectivity engineered into the bacterial homotetrameric Na+-channel NavAb; 3, 4).

Together these three publications provide important insight into how small molecule channel blockers interact with their high affinity binding domains within Ca2+ channels. They also reveal a remarkable similarity between the core structures (i.e. the voltage-sensors and the pore domains) between not only the closely related Cav1.1 and Cav3.1 channels but also their bacterial ancestor CavAb.

Moreover, although some differences exist, the binding poses of L-type Ca2+ channel blockers in the mammalian Cav1.1 and the bacterial CavAb channel are very similar. In both structures verapamil and diltiazem bind within the central cavity, compatible with their pore-blocking properties predicted in functional studies. In contrast, dihydropyridines like nimodipine and nifedipine bind within a fenestration created by adjacent transmembrane S6 helices (IIIS6 and IVS6). These fenestrations penetrate the sides of the central cavities in both voltage gated Na+– and Ca2+ channels and connect them to the surrounding lipid bilayer of the plasma membrane (5). In accordance with functional studies, these dihydropyridines must therefore inhibit L-type channels by allosterically interfering with gating rather than pore block.

These findings raise the important question about how Z944 can selectively inhibit T-type Ca2+ channels. This drug is currently in clinical development for the treatment of epilepsy and neuropathic pain. It appears that Z944 bound to Cav3.1 combines the binding modes of both pore blockers and allosteric blockers. Its phenyl ring projects into the fenestration between repeats II and III (similar to nifedipine binding), whereas the other end of the molecule projects down through the central cavity to the intracellular mouth of the pore. A prominent interaction was found with lysine-1462, a residue unique for T-type channels. Its mutation to L-type residues reduced drug sensitivity in functional studies suggesting that it is one of the determinants for T-type selectivity.

Taken together the accumulating structural insight into drug binding and modulation will greatly facilitate structure-guided drug discovery. This may lead to new oral subtype-selective blockers of voltage-gated Ca2+ channels with therapeutic potential for the treatment of epilepsy (T-type), pain (T-type, N-type), tremor (T-type), primary aldosteronism (Cav1.3 L-type), progression of Parkinsons disease (Cav1.3, R-type) or rare neurodevelopmental disorders (Cav1.3 L-type) (6,7).

Comments by Jörg Striessnig, University of Innsbruck, Chair for NC-IUPHAR Subcommitee for Voltage-gated calcium channels, Liaison for NC-IUPHAR subcommittees on Voltage-gated ion channels

  1. Zhao, Y., Huang, G., Wu, Q., Wu, K., Li, R., Lei, J., Pan, X., and Yan, N. (2019). Cryo-EM structures of apo and antagonist-bound human Cav3.1. Nature. 576, 492–497 [PMID: 31766050].
  2. Zhao, Y., Huang, G., Wu, J., Wu, Q., Gao, S., Yan, Z., Lei, J., and Yan, N. (2019). Molecular Basis for Ligand Modulation of a Mammalian Voltage-Gated Ca2+ Cell 177, 1495-1506 [PMID: 31150622].
  3. Tang, L., El-Din, T.M.G., Swanson, T.M., Pryde, D.C., Scheuer, T., Zheng, N., and Catterall, W.A. (2016). Structural basis for inhibition of a voltage-gated Ca2+ channel by Ca2+ antagonist drugs. Nature 537, 117–121 [PMID: 27556947].
  4. Tang, L., Gamal El-Din, T.M., Lenaeus, M.J., Zheng, N., and Catterall, W.A. (2019). Structural Basis for Diltiazem Block of a Voltage-Gated Ca2+ Mol. Pharmacol. 96, 485–492 [PMID: 31391290].
  5. Payandeh, J., Scheuer, T., Zheng, N., Catterall, W.A., 2011. The crystal structure of a voltage-gated sodium channel. Nature 475, 353–358 [PMID: 21743477].
  6. Pinggera, A., and Striessnig, J. (2016). Cav 1.3 (CACNA1D) L-type Ca2+ channel dysfunction in CNS disorders. J. Physiol. (Lond.) 594, 5839–5849.
  7. Benkert, J., Hess, S., Roy, S., Beccano-Kelly, D., Wiederspohn, N., Duda, J., Simons, C., Patil, K., Gaifullina, A., Mannal, N., et al. (2019). Cav2.3 channels contribute to dopaminergic neuron loss in a model of Parkinson’s disease. Nat. Commun. 10, 5094. doi:10.1038/s41467-019-12834-x
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Hot Topics: Deciphering the crystal structure of the leukotriene receptor CysLT2 opens up for improved therapeutics

Leukotrienes are lipid mediators of inflammation, initially recognized for their role in asthma, but also having potent effects in for example cardiovascular and neurological diseases as well as in cancer. The initial pharmacological classification of leukotriene receptors based on antagonist selectivity in smooth muscle was shown to be valid at the gene and protein levels. Following the recent description of the CysLT1 receptor structure, Gusach et al. now describe the crystal structures of the CysLT2 receptor in complex with dual CysLT1/CysLT2 receptor antagonists (1). This is an important advancement, since it allows to reveal specific characteristics of the two CysLT receptors in terms of ligand recognition and subtype selectivity. In this context, differences in the intracellular helix 8 emerge as particular subtype determinants, which may affect G-protein regulation and β-arrestin binding. The study by Gusach et al. (1) further provides structural insights into the changes in ligand binding and downstream signaling by CysLT2R disease-related single nucleotide polymorphisms (SNP) associated with asthma and cancer. The deciphering of the crystal structures for the CysLT1 and CysLT2 receptors will open up for the design of CysLT receptor antagonists with improved affinity/efficacy or subtype selectivity profiles. The insights into the CysLT receptor genome-structure-function relations may facilitate the prediction of disease associations and potentially further expanding the therapeutic indications of CysLT receptor antagonists.

Comments by Magnus Bäck, MD PhD, (@TransCardio), Karolinska Institutet and University Hospital, Stockholm, Sweden; Chairman NC-IUPHAR subcommittee on Leukotriene Receptors

(1) Gusach, A., Luginina, A., Marin, E. et al. Structural basis of ligand selectivity and disease mutations in cysteinyl leukotriene receptors. Nat Commun 10, 5573 (2019) doi:10.1038/s41467-019-13348-2 [PMID:31811124]

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Hot Topics: New crystal structure of the muscarinic M5 receptor completes the set

Muscarinic receptors consist of 5 G protein-coupled receptors which along with nicotinic ion channels mediate the effects of acetylcholine. Despite years of research on the role of muscarinic receptors in the brain and periphery the Muscarinic M5 receptor has stood out in contrast to M1 -M4 as one which we know little about. The M5 receptor has a unique and unusual distribution in the brain being enriched in mid brain dopamine neurons and in the cerebellum. A lack of selective chemical tools has hampered our ability to study the function of the receptor. Now Vuckovic et al. [1] have solved the X-ray crystal structure of the M5 receptor in complex with the non-selective antagonist tiotropium in the presence of a conformational stabilizing mutation. Since tiotropium was also used to solve the structure of several other muscarinic receptors a direct comparison can be made across the subtypes which will enable the design of selective tool compounds. Significant progress has been made in utilizing crystal structures to design selective orthosteric and allosteric ligands at this family of receptors. This final structure completes the set of muscarinic receptor structures solved and hopefully will pave the way to further understanding of the biology of M5 and its role in disorders such as drug addiction and depression.

Comments by Fiona H. Marshall, Discovery Research UK, MSD (@aston_fm)

(1) Vuckovic Z et al. (2019). Crystal Structure of the M5 Muscarinic Acetylcholine Receptor. Proc Natl Acad Sci USA, DOI: 10.1073/pnas.1914446116. [PMID: 31772027]

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Database Release 2019.5

Our fifth and final database release of 2019 has been made and includes a number of minor content and website updates. Crucially, it comes at a time when we have just published our most recent database update in Nucleic Acids Research:

Armstrong JF, Faccenda E, Harding SD, Pawson AJ, Southan C, Sharman JL, Campo B, Cavanagh DR, Alexander SPH, Davenport AP, Spedding M, Davies JA; NC-IUPHAR. (2019) The IUPHAR/BPS Guide to PHARMACOLOGY in 2020: extending immunopharmacology content and introducing the IUPHAR/MMV Guide to MALARIA PHARMACOLOGY. Nucl. Acids Res. pii: gkz951. doi: 10.1093/nar/gkz951 [Epub ahead of print]. [Full text]

and we have seen the publication of the 2019/20 Concise Guide to Pharmacology:

Alexander SPH, Kelly E, Mathie A, Peters JA, Veale EL, Faccenda E, Harding SD, Pawson AJ, Sharman JL, Southan C, Buneman OP, Cidlowski JA, Christopoulos A, Davenport AP, Fabbro D, Spedding M, Striessnig J, Davies JA; CGTP Collaborators. (2019) The Concise Guide to PHARMACOLOGY 2019/20 Br J Pharmacol. 176 S1: S1-S493. [Table of Contents]

JWS-SH-BJP-ConciseGuide-01-Pharmacology-1120x600px-BJP

Content Updates

Other Updates

Ligand list download

  • A new download button has been added to the ligand list pages. This enables users to download the specific ligand set they are viewingfor downloading specific sets of ligands (see image below).

Screenshot from 2019-11-15 10-04-37

PlasmoDB links

  • The IUPHAR/MMV Guide to Malaria Pharmacology now includes direct links from targets to PlasmoDB. The detailed target pages now clearly display the PlasmoDB accession for the target. The links are also included in the summary view of the target.

Upcoming presentations at:

We will be presenting on the Guide to Immunopharmacology and the Guide to Malaria Pharmacology at the following meeting over November and December

 

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Hot Topics: 3D structure of the full-length P2X7 receptor provides insight into factors controlling agonist potency and receptor desensitisation

P2X receptors are ligand-gated cation channels for which ATP is the endogenous orthosteric agonist. Seven P2X subunits have been identified and they form trimers to produce at least twelve different receptor subtypes. The tertiary structure of several subtypes have been reported, but they all lack clear information on the conformation of the N- and C-terminal cytoplasmic domains because of the truncated constructs used and the flexibility of these domains. Now, McCarthy et al., (1) report single-particle cryo-EM images of the full-length rat P2X7 receptor in both apo (closed pore) and ATP-bound (open pore) states, which suggest why the affinity of this receptor for ATP is low, indicate how cysteine residues in the C-terminal control desensitisation and reveal a surprising guanine nucleotide binding site in the C-terminal.

The potency of ATP at P2X7 is three orders of magnitude lower than at other P2X subtypes. Whilst these new structures show some differences between the orthosteric ATP binding pocket of P2X7 and P2X3 receptors, these are unlikely to explain the great difference in ATP potency. Notably, however, the entrance to the pocket is much narrower in P2X7 (<11A° orifice) than in P2X3 (17A° orifice) receptors. This and any protein flexibility that opens and closes the entrance would decrease the time ATP spends in the binding pocket, so decreasing its affinity.

A unique feature of P2X7 receptors is an 18-amino acid long cytoplasmic region at the end of TM2 (named the C-cys anchor by the authors) that is cysteine-rich and which links TM2 to the cytoplasmic cap, a structural domain formed by N- and C-terminal residues that determines the rate at which P2X receptors desensitise. The present report shows that the C-cys anchor contains at least four cysteine residues and one serine residue that are palmitoylated and that the aliphatic chains extend into the plasma membrane, anchoring the receptor to the membrane. The authors speculate that this could keep the cytoplasmic cap in place and so limit P2X7 desensitisation. Consistent with this, the receptor, which is normally non-desensitising in the presence of ATP, desensitised rapidly and fully when the C-cys anchor was deleted or the cysteine residues removed by mutation.

A further unique feature of P2X7 receptors compared with other subtypes is the long C-terminal (~200 residues), which the authors term the cytoplasmic ballast. The images show for the first time that each receptor has three globular, wedge-shaped cytoplasmic ballasts, each of which hangs beneath the TM domain of an adjacent subunit. Intriguingly, each cytoplasmic ballast contains a dinuclear zinc ion complex and a high-affinity guanosine nucleotide binding site, the functions of which are unclear.

The P2X7 receptor is of particular therapeutic interest because it is cytotoxic due to its ability to activate the NLRP3 inflammasome and release pro-inflammatory cytokines. This study substantially extends our knowledge and understanding of its pharmacological and biophysical properties and forms the basis of further potential experiments designed to fully characterise how it functions.

Comments by Dr. Charles Kennedy, Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde

(1) McCarthy et al. (2019). Full-length P2X7 structures reveal how palmitoylation prevents channel desensitization. Cell. https://doi.org/10.1016/j.cell.2019.09.017. [ScienceDirect: View Article]

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