Hot topic: A new research avenue investigating mitochondrial GPCR biology

As one of the first propositions for GPCRs being present in mitochondrial membranes, a recent report from Robert Friedlander and colleagues [1] follows on from previous work characterising synaptic and extrasynaptic mitochondria in human cortex (post-mortem samples) and their role in neuroprotection. This work, if reproduced, opens up new vistas, and has many implications for neurodegenerative diseases. Taken together, Suofu et al. show that melatonin is synthesised in mitochondria, that MT1 receptors are present in mitochondrial membranes, and that MT1 receptor stimulation reduces cytochrome c and caspase secretion caused by calcium overload. The authors propose that this is a mechanism for the neuroprotective effects of melatonin in hypoxic-ischaemic brain injury in neonatal and in models of Huntington’s disease, where there is mitochondrial impairment.

Comments by Michael Spedding, Secretary General, IUPHAR, and CEO, Spedding Research Solutions SARL, France

(1) Suofu Y et al. (2017). Dual role of mitochondria in producing melatonin and driving GPCR signaling to block cytochrome c release. Proc Natl Acad Sci U S A., pii: 201705768. doi: 10.1073/pnas.1705768114. [Epub ahead of print] [PMID:28874589]

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Hot topic: Crystal structure of LPA6, a receptor for lysophosphatidic acid, at 3.2A

Lysophospholipids (LPs) have myriad roles as extracellular signals that activate cognate G protein-coupled receptors (GPCRs) (2). LPs for which receptors have been reported include lysophosphatidic acid (LPA) (receptors: LPA1-6), sphingosine 1-phosphate (S1P1-5), lysophosphatidyl serine (LPS1-3, 2L (2L is a pseudogene in humans)) and lysophosphatidyl inositol/glucose (LPI/LPG), all of which are Class A GPCRs. Of these 15 LP receptors, crystal structures of two have been previously reported for S1P1 (2.8-3.35A) (3) and LPA1 (2.9-3.0A) (4) both of which utilized human cDNA sequences bound in the presence of antagonists. The new structure (1), from the laboratories of Junken Aoki and Osamu Nureki, elucidates a zebrafish receptor – with 80% amino acid similarity to human LPA6, in the transmembrane (TM) region – in the absence of a ligand, which nonetheless crystalized. This contrasts with the prior 2 antagonist-bound human structures. All 3 receptors were chimeric proteins stabilized by T4-lysozyme (S1P1 and LPA6) or thermostabilized apocytochrome b562RIL (LPA1) fused to the 3rd intracellular loop, but all were capable of responding to native ligands.

Key features of LPA6 included a surprisingly large distance between TM4 and 5, which suggests lateral entry of LPA via membrane translocation into the LPA6 binding pocket. Such a mechanism contrasts with that of LPA1 in which TM1 and 7 distances are comparatively small, and whose structure includes a barrel opening flexibly covered by an unstabilized N-terminal helix that contrasts with a stabilized helix in S1P1 that could inhibit ligand entry from extracellular space. LPA1’s structure is further consistent with LPA entry from the extracellular environment that could include its biosynthetic enzyme, autotaxin. By comparision, both S1P1 and LPA6 – despite being of distinct gene sub-families (EDG and P2Y, respectively) – show receptor entry of ligands from within the membrane plane, suggesting parallel evolution of membrane access for these gene sub-families. LPA6 prefers unsaturated LPAs (e.g., 18:2) that appear to enter a hydrophobic cleft and central cavity binding site that supports unsaturated LPA species based upon docking models. Modeling also supports LPA-binding that produces a shift of TM6 and 7 to allow more favorable interactions with LPA’s phosphate headgroup. Membrane access of LPA into LPA6 is further supported by actions of the phospholipase PA-PLA1α that was shown to increase membrane LPA without extracellular secretion, thus providing membrane ligand that could translocate into LPA6.

Comments by Jerold Chun, MD, PhD, Professor & Senior Vice President, Neuroscience Drug Discovery, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA

  1. Taniguchi, R. et al. (2017) Structural insights into ligand recognition by the lysophosphatidic acid receptor LPA6. Nature, 548, 356-360, doi:10.1038/nature23448. [PMID:28792932]
  2. Kihara, Y. et al. (2014) Lysophospholipid receptor nomenclature review: IUPHAR Review 8. Br J Pharmacol, 171, 3575-3594, doi:10.1111/bph.12678 . [PMID:24602016]
  3. 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]
  4. Chrencik, J. E. et al. (2015) Crystal Structure of Antagonist Bound Human Lysophosphatidic Acid Receptor 1. Cell, 161, 1633-1643, doi:10.1016/j.cell.2015.06.002 . [PMID:26091040]
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Hot topic: FZD6 dimers dissociate after stimulation – briefly

GPCRs of all classes are widely thought to form homodimers, heterodimers and higher-order oligomers. The functional significance of dimerization is well understood for Class C receptors but less certain for the other GPCR classes, including the rather unconventional class F or Frizzled (FZD) receptors. Although the relationship between receptor activity and quaternary structure is often unclear, across classes it is generally found that ligand binding does not dramatically influence dimerization. A recent report by Gunnar Schulte and his colleagues suggests that in this respect class F receptors may once again be somewhat different [1]. Using an impressive combination of live-cell imaging, biochemical and modeling techniques the group presents evidence that FZD6 forms relatively stable dimers that dissociate when stimulated with the activating ligand WNT-5A. Remarkably, FZD6 protomers reassociate at the cell surface after 20 minutes of continuous stimulation, a timing which coincides with termination of ERK1/2 phosphorylation. Taken together with previous results from the Schulte group [2] the data are consistent with a model where FZD6 dimers are constitutively associated with G proteins and the phosphoprotein Disheveled (DVL) in an inactive state complex that must dissociate in order to generate downstream signals. Although it remains to be seen how representative this model will be for other GPCRs, including other class F receptors, the report sets an important standard for studies aimed at linking receptor activity and quaternary structure.

Comments by Nevin A. Lambert, PhD, Department of Pharmacology and Toxicology Medical College of Georgia, Augusta University, USA

[1] Petersen, J., Wright, S.C. et al. (2017) Agonist-induced dimer dissociation as a macromolecular step in G protein-coupled receptor signaling. Nat Commun. 8(1):226.  [PMID: 28790300]

[2] Kilander, M.B.C., Petersen, J. et al. (2014) Disheveled regulates precoupling of heterotrimeric G proteins to Frizzled 6. FASEB J. 28(5):2293-305. [PMID: 24500924]

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Hot topics: A cryptic binding pocket in K2P2 exposes new avenues for drug development.

The TREK subfamily of K2P channels (K2P2, K2P4 and K2P10) pass background potassium currents that modulate the excitability of neuronal cells and cardiac myocytes. In recent years, these channels have received significant attention as potential drug targets. This is in part because of their proposed roles in the regulation of nociception, analgesia, anesthesia and depression and, also because they act as polymodal signal integrators for physiological influences as diverse as temperature, membrane tension, phosphorylation, and phospholipids [1-3]. However, despite accelerating progress, including atomic resolution structures of two TREK subfamily members [4,5] , and K2P1 [6], a deeper appreciation of how K2P channel structure-function relationships, including gating mechanics, relate to physiology and disease remains hampered by a paucity of specific blockers and activators. In an elegant new study, Lolicato and colleagues from the Minor lab highlight the synergistic power of combining structural and functional approaches to reveal new insights into the operation of membrane proteins and unveil a new avenue for the development of TREK-channel pharmacology [7].
Lolicato et al., describe crystal structures of mouse K2P2 (TREK1) in complex with a novel K2P2-specific activator (ML335), and a K2P10 activator (ML402) [7]. ML335 and ML402 occupy a previously unidentified binding site—the K2P modulator pocket. Like all K2P channels, K2Ps 2, 4 and 10 are composed of two subunits, each with two pore domains and can assemble as homomers or heterodimers. Given the bilateral nature of the K2P structure, each channel will have two modulator pockets. The modulator pocket is located between the P1 and M4 helical domains in each subunit where residues conserved among TREK subfamily channels interact with the ML335 and ML401 via cation-π and π-π interactions.

While the activity of most ion channels is controlled by multiple gates, experimental evidence has accumulated to support the idea that K2P channels use a single C-type gate at the outer pore which controls ionic flux by the mechanics of the selectivity filter for potassium ions [8-10]. Numerous studies suggest that the unique architecture of K2P channels routes diverse regulatory signals to the C-type gate to control channel activity [9,11-13]. Thus, operation of the C-type gate is directly sensitive to changes in the permeant ion [11,13] and indirectly influenced by various K2P channel regulators that interactions with domains that in-turn impact the C-type gate [12-16]. The modulator pocket described lies behind the selectivity filter. Functional studies show that ML335 holds the pocket in an open conformation and thereby, activates the channel by stabilizing the C-type gate of K2P2 [7]. Because modulator pocket activators appear to be sufficient to open K2P2 channels, the findings suggest that this previously unappreciated, druggable site can be leveraged for the development of novel channel gating-modulators with potential utility as analgesics, anesthetics or neuroprotective agents.

Comments by Leigh D. Plant, Ph. D. (Research Associate Professor, School of Pharmacy, Northeastern University)

[1] Goldstein, S. A. et al. (2001). Potassium leak channels and the KCNK family of two-P-domain subunits. Nat Rev Neurosci 2, 175-184. [PMID: 11256078</a].

[2] Enyedi, P. & Czirjak, G. (2010). Molecular background of leak K+ currents: two-pore domain potassium channels. Physiol Rev. 90, 559-605. [PMID: 20393194].

[3] Honore, E. (2007). The neuronal background K2P channels: focus on TREK1. Nat Rev Neurosci. 8, 251-261. [PMID: 17375039].

[4] Brohawn, S. G., del Marmol, J. & MacKinnon, R. (2012). Crystal structure of the human K2P TRAAK, a lipid- and mechano-sensitive K+ ion channel. Science, 335, 436-441. [PMID: 22282805].

[5] Dong, Y. Y. et al. K2P channel gating mechanisms revealed by structures of TREK-2 and a complex with Prozac. Science, 347, 1256-1259. [PMID: 25766236].

[6] Miller, A. N. & Long, S. B. (2012) Crystal structure of the human two-pore domain potassium channel K2P1. Science, 335, 432-436. [PMID: 22282804].

[7] Lolicato, M. et al. (2017). K2P2.1 (TREK-1)-activator complexes reveal a cryptic selectivity filter binding site. Nature, 547, 364-368. [PMID: 28693035].

[8] Zilberberg, N., Ilan, N. & Goldstein, S. A. (2001). KCNKØ: opening and closing the 2-P-domain potassium leak channel entails “C-type” gating of the outer pore. Neuron, 32, 635-648. [PMID: 11719204].

[9] Piechotta, P. L. et al. (2011). The pore structure and gating mechanism of K2P channels. EMBO J, 30, 3607-3619. [PMC: PMC3181484].

[10] Schewe, M. et al. (2016). A Non-canonical Voltage-Sensing Mechanism Controls Gating in K2P K(+) Channels. Cell 164, 937-949. [PMID: 26919430].

[11] Cohen, A., Ben-Abu, Y., Hen, S. & Zilberberg, N. (2008). A novel mechanism for human K2P2.1 channel gating. Facilitation of C-type gating by protonation of extracellular histidine residues. J Biol Chem, 283, 19448-19455. [PMID: 18474599].

[12] Bagriantsev, S. N., Clark, K. A. & Minor, D. L., Jr. (2012). Metabolic and thermal stimuli control K(2P)2.1 (TREK-1) through modular sensory and gating domains. EMBO J, 31, 3297-3308. [PMC: PMC3411076].

[13] Bagriantsev, S. N. et al. (2011). Multiple modalities converge on a common gate to control K2P channel function. EMBO J, 30, 3594-3606. [PMID: 21765396].

[14] Chemin, J. et al. (2005). A phospholipid sensor controls mechanogating of the K+ channel TREK-1. EMBO J, 24, 44-53. [PMID: 15577940].

[15] Murbartian, J., Lei, Q., Sando, J. J. & Bayliss, D. A. (2005). Sequential phosphorylation mediates receptor- and kinase-induced inhibition of TREK-1 background potassium channels. J Biol Chem, 280, 30175-30184. [PMID: 16006563].

[16] Honore, E., Maingret, F., Lazdunski, M. & Patel, A. J. (2002). An intracellular proton sensor commands lipid- and mechano-gating of the K(+) channel TREK-1. EMBO J, 21, 2968-2976. [PMID: 12065410].

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Anti-infective pilot entries

GtoPdb has been traditionally focused on the pharmacology associated with human diseases (i.e. we have not been funded to cover anti-infectives).  In 2017 we have been exploring possible funding opportunities to extend into  expert curation of anti-infectives, particularly in the light of the antibiotic resistance threat and expanding drug discovery efforts for neglected tropical diseases (NTDs).  Consequently, for this release we have added a selection of entries as a proof of concept for how well our current data model would accommodate these new types of relationship mappings “off the bat”.  These are now under a new  category of “Anti-infective targets“.  A snapshot of the six new entries is show below.


We curated three antimalarials,  two antivirals and one antibiotic, some of which have the ligand in PDB entries. By and large,  this pilot was successful and we would be pleased to get feedback from interested parties.  A number of technical challenges were encountered  but most of these were “domain inherent” rather than GtoPdb data model issues per se.  Examples include the sub-species and strain multiplexing of the target sequences. This results in having to map the authors described ligand activity data to TrEMBL entries (sometimes with equivocalities as to the exact isolate sequence) rather than to the deeper annotated Swiss-Prot entries avaialble for some reference pathogen proteomes with completed gene naming.  We also ran in to the multi-enzyme “string of pearls” problem where large polypeptides encode for multiple  functional domains, more than one of which that can be (or have been) targeted by different inhibitors.  Classically, this is the case for the viral polyprotein precursor proteins but in this set also for the polyketide synthase entry shown below.


This was derived from a recent 2017 Cell paper  “Development of a Novel Lead that Targets M. tuberculosis Polyketide Synthase 13” (PMID 28669536). Should we be sucessful in being resourced to expand in this domain we can already envisage tweaks to our existing data model and curation processes that could address some these domain-specific challenges.  For example,  we can specify ligand-targeted domains not only by InterPro coordinates in UniProt entries but also by getting the TrEMBL entries “promoted” to Swiss-Prot to enhance the domain annotation cross-references.


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New source cross-references in release 2017.5

(minor updates 15 Sep 2017)

The statistics of content are presented as usual in the release notes and The Guide to IMMUNOPHARMACOLOGY has a separate update.  This post describes changes and updates to other resources we provide links to, that have been introduced in this release cycle.  More detail will be provided in the help pages (and feedback on any of them is welcome) but the outlines are as follows;

Extra links for ligands. The new connectivity applies to those that have chemical structures (i.e. SMILES strings for mostly small molecules but also peptides up to ~ 50 to 60 residues and a few oligonucleotide drugs), which represents 6821 ligands in GtoPdb. Links have now been rationalised by introducing InChIKey call-outs to UniChem at the EBI.  This resource, currently containing over 150 million indexed chemical structures from 37 sources (including our own), many of which we had hitherto individually curated links for.  In essence, UniChem “looks after” comprehensive cross-mappings between these sources via a regular and precise automated process. We can consequently rely on presenting these links for our own entries. This is because we have selected and curatorially checked (i.e. locked-down) our own structural assignments, including for our PubChem submissions.  By clicking the UniChem link users can now quickly navigate to complementary sources  such as  DrugBank, ChEBI, HMDB, BindingDB, ChEMBL, PDBe SureChEMBL (patents) and others. Note this is analogous to the Google InChIKey call out we already introduced for our ligands some time ago. There is some overlap in the result sets but note the Google search will find different chemistry sources (including ChemSpider entries, usualy uppermost in the Google rankings) that are not currently indexed by UniChem.

The Human Protein Atlas (HPA) team have increased their profile recently,  not only by becoming one of the European ELIXIR core resources but also because of a major new extension in the form of a Pathology Atlas with a focus on human cancer.  We have also had contacts with the team.  Consequently,  we selected this as as a new outlink from our human protein entries (2839 target links and 353 ligand links) as an excellent first-stop shop for tissue and cell line expression patterns as well as intracellular distributions.  In terms of utility it is important to note that HPA offers the best of both worlds by integrating three sources of high-throughput mRNA transcript profiling in addition to direct antibody detection of the protein.

CATH/Gene3D. As you may have been noticed we have increased our protein structure connectivity in 2017 including our SynPharm drug-responsive protein sequences resource (see below).  There are many user utilities for the increase in structural data, including the impressive acceleration of ligand binding sites resolved in new GPCR structures. CATH is a classification of PDB protein structures grouped by protein domains into superfamilies that have diverged from a common ancestor. Users are encouraged to take a look at the  features of CATH for their own exploitation. These include tracking the deep phylogeny of pharmacological targets (that have structures) where this is difficult to detect on the basis of sequence similarity alone. The current version of GtoPdb includes 1634 target links to CATH (which is lower than the total because not all protein families have 3D structural representation, yet), and 230 peptide ligands  (Sep 2017 update CATH is also now European ELIXIR core resources).

synPHARM was originally set-up to provide synthetic biologists with tools to discover sequences that could be modulated by known ligands from GtoPdb which could be transferred to synthetic proteins in order to confer drug control. synPHARM combines structural information from the Protein Data Bank with information on ligand binding from GtoPdb to produce a database of ligand binding sequences. As such, it is a useful resource for 3D ligand binding information. We have now added links from GtoPdb target and ligand pages to structures in synPHARM.

IUPHAR Pharmacology Education Project (PEP). PEP is a new IUPHAR initiative to provide free access to education and training resources in pharmacology. We have added links from 673 ligand and drug pages to background information in PEP, for further information on drug action and clinical use.

RCSB Protein Data Bank (RCSB PDB). Although not strictly new, it’s worth pointing out that the current rate of reporting new structures of ligands bound to targets means the number of links to the PDB via ligand entries has increased significantly over recent releases. The number of our PDB ligand links now stands at 1337, based on exact InChIKey matches. In addition, many of the GtoPdb ligands are represented in the PDB as alternative isomeric forms. Note also there are occasions where the PubChem MMDB CID assignment does not exactly match the PDB ligand structure.  In both these cases we add cross-pointers in the ligand comment sections.

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Database release 2017.5

The 5th IUPHAR/BPS Guide to PHARMACOLOGY database release of 2017 includes updates to several target families, and new targets and ligands added, focusing on those relevant to immunopharmacology. We also announce a new organisation for ligand families and groups. This update also includes the beta v2.0 release of the IUPHAR Guide to IMMUNOPHARMACOLOGY portal taking into account early feedback from our beta testers. Eagle-eyed users may have noticed a new homepage layout for GtoPdb, which has been reorganised to highlight important new features at the top of the page, with quick links to the main database pages on the left, and news items and publications below.

Target and ligand updates

Ligand families

We have introduced a new organisation of peptide ligands into families. This can be reached via a link from the “Ligands” submenu of the main navigation menu. This started with the aim of grouping related peptide sequences together into families to aid discoverability and allow us to add comments and references pertaining to the family as a whole. We have also experimented with grouping together some other types of ligands (such as the Immune checkpoint modulators) linked by their mechanism of action (although not a family in the phylogenetic sense). Feedback on this new organisation is welcome.

Ligand activity graphs

Continuing on from previous updates (releases 2017.2 and 2017.4), where we described the addition of graphs to visualise ligand activity data for targets across species using data from GtoPdb and the med-chem database, ChEMBL, we have now extended this feature to all ligands in GtoPdb with quantitative activity data at targets, even where the ligands do not have data in ChEMBL. There will  also be cases where the GtoPdb curators just haven’t yet identified the ligand in ChEMBL, in particular peptides can be difficult to search for because of naming differences and lack of standard chemical structure descriptors.

Expanded database cross-links

From time to time we internaly review the databases that we cross-link to and from, to make sure they are current and useful. During an iteration of this process within this release cycle we introduced several new resources that have value for users. These changes are explained in a separate post.


This update also sees the release of the beta v2.0 of the new Guide to IMMUNOPHARMACOLOGY portal. This is a Wellcome Trust-funded extension to the existing database, aiming to improve data exchange between immunology and pharmacology. Read the release notes and technical update here. We are grateful for all the feedback received so far and welcome continued comments and bug reports as we further develop the new data and portal.

Database content and statistics

For the full statistics on release 2017.5 please see the about page on the GtoPdb site. In summary, there are now 15,281 curated binding constants between 2825 targets and 8978 ligands. (N.B. for various reasons, not all those targets and ligands have  quantitative binding data; in the third table below the current number of human targets with quantitative data is 1431.)


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