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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Official Launch of the Guide to MALARIA PHARMACOLOGY

gtommv_bannerWe are delighted to announce the first full public release of the IUPHAR/MMV Guide to MALARIA PHARMACOLOGY (abbreviated to GtoMPdb). This new web resource, designed specifically for malaria pharmacology, has been developed as a joint initiative between the International Union of Basic and Clinical Pharmacology (IUPHAR) and the Medicines for Malaria Venture (MMV). It has been constructed as an extension to the parent Guide to PHARMACOLOGY database (GtoPdb) and incorporates new pharmacological content, including molecular targets in the malaria parasite and interaction data for ligands with antimalarial activity. In addition, a dedicated portal has been developed, in consultation with key opinion leaders in malaria research, to provide quick and focused access to these new data.

Screen Shot 2019-09-26 at 15.32.20The GtoMPdb portal homepage

A more detailed introduction to the project and a chronicle of technical developments can be found in our previous blog posts.

This initiative has enriched the GtoPdb and it is hoped will foster innovation by providing, in a single expert-curated database, results from antimalarial drug discovery programmes and the scientific literature. Following the lastest database release (2019.4), the Antimalarial targets family and the Antimalarial ligands family were updated and now contain a total of 30 P. falciparum (3D7) targets and 72 ligands annotated with antimalarial activity. The GtoMPdb has maintenance funding, ensuring new data curation will continue until June 2020.

We would like to take this opportunity to thank everyone involved in the project and for the helpful feedback we received during beta-testing of the portal. If you have any further feedback or queries about the resource please contact enquiries@guidetopharmacology.org

This project is supported by a grant awarded to Professor Jamie Davies at the University of Edinburgh by Medicines for Malaria Venture (MMV).

 

 

 

 

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Database Release 2019.4

We are very pleased to announce our a new release of the IUPHAR/BPS Guide to Pharmacology. This version (2019.4) is the fourth this year and includes the first full-release of the IUPHAR/MMV Guide to Malaria Pharmacology.

Content Updates

GtoPdb now contains over 9,800 ligands, with around 7,450 having quantitative interaction data to biological targets. 1,442 of the ligands are approved drugs. The database contains over 1,700 human targets with curated interactions, with just over 1,500 of these having quantitative data. Full stats can be found on our About Page.

Here’s a brief summary of some of main curatorial updates:

  • The Lysyl oxidases family has been added to allow the GtoPdb to capture advance in medicinal chemistry and development of lysyl oxidase inhibitors. Lysyl oxidases are extracellular enzymes that are vital for cross-linking fibrillar elastin and collagens and for extracellular matrix stabilisation. LOX and LOXL2 are implicated in fibrosis, tumourigenesis, and metastasis, and are subsequently molecular targets for cancer drug discovery.
  • We have cross-referenced GtoPdb ligands against the World Health Organization (WHO) Model List of Essential Medicines, and now include this subset as a specific ligand category on our ligand list page. This classification is also displayed on ligand summary pages. Currently, 193 ligands in GtoPdb are on the WHO list.

Guide to Malaria Pharmacology (GtoMPdb)

We are delighted to officially launch the first full public release of the IUPHAR/MMV Guide to Malaria Pharmacology. GtoMPdb has been constructed in partnership with the Medicines for Malaria Venture (MMV), an organization dedicated to identifying, developing and delivering new antimalarial therapies that are both effective and affordable. This is in response to the global challenge of over 200 million cases of malaria and 400 000 deaths worldwide, with the majority in the WHO Africa Region.  It provides new pharmacological content, including molecular targets in the malaria parasite and interaction data for ligands with antimalarial activity.  We have pioneered curation of data from assays screening compounds against the whole organism, used routinely in antimalarial drug discovery.

A dedicated portal has been developed to provide quick and focused access to these new data and we have added direct links on the main GtoPbd home page.

home6

GtoPdb home page with new menu bar links to GtoMPdb.

In this database release these are the recent advancements made in the GtoMPdb.

  • The Antimalarial targets family and the Antimalarial ligands family have been updated, giving a total of 30 P. falciparum (3D7) targets and 72 ligands tagged as antimalarial in the database.
  • We have fixed the display of interactions to avoid duplicate rows – now data for a single target, ligand and species will appear together.

You can read more about the launch at this blog post.

Other Updates

Immunopharmacology content statistics

On the immunopharmaoclogy help page we have added a dynamic list of database content stats.

External links in Europe PMC

The GtoPdb has recently been included in the External Links service at Europe PMC (EPMC)  (https://europepmc.org/LabsLink). On EPMC pages, links to target and ligand entries have been added to the papers curated by GtoPdb that include a quantitative description of the ligand-target interaction. It is possible to retrieve all these references at EMPC by running an “Advanced Search” and selecting “IUPHAR/BPS Guide to Pharmacology” from the “External Links” drop-down list (LABS_PUBS:”1969″) as the cross-reference query. This currently gives 1,729 results, which can be further combined as Boolean-type queries against all other types of EPMC indexing including Bibliographic Fields, Filters, Data Links, External Links and Annotations.

empc

External Links for PMC:6452685 – showing links back to GtoPdb targets and ligands.

Bioschemas

We are also working with Bioschemas (http://bioschemas.org/) (35) to add schema.org semantic mark-up to GtoPdb, which will make it simpler for search engines to index the website. Our current focus is on implementing mark-up on all ligand summary pages, including properties from the Bioschemas MolcularEntity profile (https://bioschemas.org/specifications/drafts/MolecularEntity). A first version of the ligand mark-up has been added, but it remains a work in progress as we seek to refine it.

Posted in Database updates, Guide to Immunopharmacology, Guide to Malaria Pharmacology, Technical