GPR139 is an orphan class A G protein-coupled receptor found mainly in the central nervous system. It has its highest expression in the striatum and hypothalamus, regions regulating locomotion and metabolism, respectively, and it has therefore been suggested as a potential target for Parkinson’s disease and metabolic syndrome. Surrogate ligands have been published by Lundbeck A/S [1], Jansen R&D [2], Takeda Pharmaceuticals [3], as well as the University of Copenhagen (Gloriam group). In a new publication, the latter group describe the first combined structure-activity relationships of all surrogate agonist, and a common pharmacophore model for future ligand identification and optimization [4].

The physiological agonist of GPR139 is still elusive. GPR139 has previously been shown to be activated by the amino acids l-tryptophan and l-phenylalanine (EC50 values of 220 μM and 320 μM, respectively) [5,6], as well as di-peptides [5]. A new publication shows that the endogenous melanocortin 4 receptor agonists; adrenocorticotropic hormone and α- and β-melanocyte stimulating hormone in the low micromolar range. In addition, a potentially novel subpeptide (from consensus cleavage site) represents the most potent putative endogenous activator, so far (EC50 value of 600 nM) [7]. Together, these results indicate that GPR139 is a likely to be a peptide receptor that could act as a secondary target for melanocortin peptides or a yet undiscovered physiological ligand.

[1] Shi, F. (2011). Discovery and SAR of a series of agonists at orphan G protein-coupled receptor 139. ACS Med. Chem. Lett. 2, 303–306. doi:10.1021/ml100293q. PMID: 24900311

[2] Dvorak, C. (2015). Identification and SAR of glycine benzamides as potent agonists for the GPR139 Receptor. ACS Med. Chem. Lett. 6, 1015–1018. 10.1021/acsmedchemlett.5b00247. PMID: 26396690

[3] Hitchchock, S. (2016). 4-oxo-3,4-dihyroI-1,2,3-benzotriazine modulators of GPR139. US Patent US2016/0145218 A1. Takeda Pharmaceutical Company Limited

[4] Shehata, M.A. (2016). Novel agonist bioisosteres and common structure-activity relationships for the orphan G protein-coupled receptor GPR139. Sci. Rep. 6, 36681. doi:10.1038/srep36681. PMID: 27830715

[5] Isberg, V. et al. (2014). Computer-aided discovery of aromatic L-α-amino acids as agonists of the orphan G protein-coupled receptor GPR139. J. Chem. Inf. Model. 54, 1553–1557. doi: 10.1021/ci500197a. PMID: 24826842

[6] Liu, C. (2015). GPR139, an Orphan Receptor Highly Enriched in the Habenula and Septum, Is Activated by the Essential Amino Acids L-Tryptophan and L-Phenylalanine. Mol. Pharmacol. 88, 911–925. doi: 10.1124/mol.115.100412. PMID: 26349500

[7] Nøhr, A.C. et al. (2016). The orphan G protein-coupled receptor GPR139 is activated by the peptides: Adrenocorticotropic hormone (ACTH), α-, and β-melanocyte stimulating hormone (α-MSH, and β-MSH), and the conserved core motif HFRW. Neurochem. Int. 102, 105–113. doi: 10.1016/j.neuint.2016.11.012. PMID: 27916541

Comments by David E. Gloriam and Anne Cathrine Nøhr Jensen (Department of Drug Design and Pharmacology, University of Copenhagen)

This year witnessed a tremendous progress in our understanding of the structure-function relationship of voltage-gated Ca²⁺ channels. This is based on the cryo-electron microscopy structure of the rabbit Cav1.1 Ca²⁺ channel complex at a nominal resolution of 3.6 Å ([1] see Hot Topics Sep 20, 2016) which is now nicely complemented by a study defining the binding domains for Ca²⁺ antagonist drugs and their molecular mechanism of action at atomic resolution [2]. The authors took advantage of their elegant previous work solving the structure of bacterial Na+ channels (NavAb) by X-ray crystallography both in a pre-open and inactivated state [3,4]. They also engineered Ca²⁺ selectivity into its selectivity filter (“CavAb”, [5]) and found high affinity inhibition by the different chemical classes of Ca²⁺ antagonist drugs similar to L-type Ca²⁺ channels. Since CavAb assembles as a tetramer, this channel also replicates the four domain structure of the pore-forming subunit of voltage-gated Ca²⁺ and Na+-channels: an excellent model to investigate the drug-channel interaction at atomic resolution was now at hand.
As predicted for L-type Ca²⁺ channel α1-subunits by photoaffinity labeling studies 25 years ago [6], dihydropyridines (DHPs, e.g. amlodipine, nimodipine) bind to an extracellularly exposed intersubunit crevice formed by neighbouring S6 helices and the P-helix of the selectivity filter. Drug binding displaces an endogenous lipid molecule in this site. Interestingly, DHP binding induces a conformational changes which breaks the fourfold symmetry of the channel. As a consequence only one molecule can occupy the channel with high affinity and the Ca²⁺ interaction with the selectivity filter also changes. This results in a higher affinity for Ca²⁺ ions revealing an intriguing mechanism of action for these drugs: rather than directly blocking the pore, they enhance Ca²⁺ affinity for the pore such that Ca²⁺ itself gets stuck in the ion conducting pathway. Therefore DHPs do the opposite of what would be intuitively expected for a “Ca²⁺ antagonist”, namely preventing Ca²⁺ interaction with the channel. Instead, they allosterically enhance Ca²⁺ binding.
In contrast, and also in agreement with photoaffinity labeling and mutational studies phenylalkylamines (PAAs, e.g. Br-verapamil) bind in the central cavity on the intracellular side of the selectivity filter also disrupting fourfold symmetry. Since it is known that PAAs access this site preferentially when the channel opens its intracellular mouth upon activation this nicely explains their known frequency dependent inhibition. Unlike DHPs these drugs bind within the pore and thus must act as pore blockers thus satisfying the term “Ca²⁺ antagonist”.
This work from the Catterall lab must be regarded as a milestone in Ca²⁺ channel research. It not only revealed the mechanism of action of one of the most prescribed classes of cardiovascular drugs but also brings us much closer to predicting structural features of new generations of Ca²⁺ antagonists with high selectivity for different isoforms of voltage-gated Ca²⁺ channels. Within the L-type Ca²⁺ channel family this could be relevant for discovering Cav1.3–selective drugs as potential therapeutics for neuroprotection in Parkinson’s disease and neuropsychiatric disorders, such as autism (7).

[1] Wu et al (2016). Structure of the voltage-gated ²⁺ channel Cav1.1 at 3.6 Å resolution.
Nature 537:191-196. [PMID 27580036]
[2] Tang et al. (2016). Structural basis for inhibition of a voltage-gated Ca²⁺ channel by Ca²⁺ antagonist drugs. Nature 537, 117–121 [PMID 27556947]
[3] Payandeh et al. (2011). The crystal structure of a voltage-gated sodium channel. Nature 475, 353–358 [PMID 21743477]
[4] Payandeh et al. (2012). Crystal structure of a voltage-gated sodium channel in two potentially inactivated states. Nature 486, 135–139 [PMID 22678296]
[5] Tang et al. (2014). Structural basis for Ca²⁺ selectivity of a voltage-gated calcium channel. Nature 505, 56–61 [PMID 24270805]
[6] Catterall and Striessnig (1992). Receptor sites for Ca²⁺ channel antagonists. Trends Pharmacol Sci 13, 256–262 [PMID 1321525]
[7] Ortner and Striessnig (2016). L-type calcium channels as drug targets in CNS disorders. Channels 10: 7–13 [PMID 26039257]

Comments by Jörg Striessnig (Department of Pharmacology and Toxicology – Institute of Pharmacy, Universität Innsbruck)

The number of alternative mRNA splice forms that map to human protein coding loci has increased to the point that nearly all proteins have such associated database records. This gives rise to the paradox that the gene build pipeline from the latest Ensembl GRCh38 reference genome assembly indicates 19,919 protein coding loci (which shrinks to 19,022 with HGNC annotation stringency) but 198,002 transcripts (i.e. nearly 10 transcripts per protein). Their is no question that a small number of these alternative splice forms, AS, (plus alternative initiations) have not only been verified to exist as proteins, have some kind of alternative biochemical functions and are also of pharmacological importance [1].  Notwithstanding, compared to the massive transcript profiling that RNAseq now provides routinely, experimentally verifying AS existence at the protein level at large scale is extremely difficult. This is because it can only be done by splice form specific antibodies, western blots detecting different size forms, top down proteomics (i.e. intact mass measurement) or the detection of alternative exon-specific trypic peptides. A recent  review [2] proposes that expanding data sets from the latter approach are consistently detecting only single quantitatively dominant protein isoforms from each locus. The provocative inference is that the vast majority of the 200K odd predicted and/or verified alternative mRNA transcripts are not actually translated into proteins.  This can be seen as an interesting methodological detection “gulph” between RNAseq and MS-proteomics.  However, their has been previous support for the “single isoform” idea on the basis of transcript data alone [3]. An ancillary conclusion from this paper, generally overlooked in terms of its significance, was that when CDS length was taken into account approximately 50% of major transcripts did not corresponding to the ‘canonical’, max-exon, transcript as annotated in Swiss-Prot. This crucial topic is further discussed in [4].

[1] Bonner, T.I. (2014). Should pharmacologists care about alternative splicing? IUPHAR Review 4. Br J Pharmacol. Mar;171(5):1231-40. doi: 10.1111/bph.12526. PMID: 24670145.

[2] Tress et al. (2016). Alternative Splicing May Not Be the Key to Proteome Complexity. Trends Biochem Sci. Sep 16. doi: 10.1016/j.tibs.2016.08.008. PMID: 27712956.

[3] Gonzàlez-Porta et al. (2013). Transcriptome analysis of human tissues and cell lines reveals one dominant transcript per gene. Genome Biol.  Jul 1;14(7):R70. doi: 10.1186/gb-2013-14-7-r70. PMID: 23815980.

[4] Will the real cannoical protein please stand up.
https://cdsouthan.blogspot.se/2016/11/will-real-canonical-proteins-please.html

Comments by Chris Southan

Endothelin is a peptide that acts via two G-protein coupled receptors. ET_A mainly causes vasoconstriction. In contrast ET_B  predominantly acts as a beneficial clearing receptor and by the release of endothelium derived relaxing factors, vasodilatation [1,2]. This paper  describes for the first time the crystal structure of  the endothelin ET_B receptor [3]. To date less than 20 structures of Family A, GPCRs (targets of nearly half of all drugs) have been solved experimentally. The number solved for small peptides ligands are limited to the opioid receptor and  the 13 amino acid neurotensin. This manuscript extends information to a much larger  21 amino acid peptide and interestingly demonstrates interaction over a substantial portion of the molecule. The authors propose a model whereby the N-terminal tail and the ECL2 β-sheet of ET_B together form a lid-like architecture that covers the orthosteric pocket, predicted to form a very stable complex. This provides one structural explanation for the unusual property of ET-1  in causing long lasting responses. Mutations in ET_B in receptors can result in Hirschsprung disease in humans, characterized by an absence of enteric ganglia in the distal colon and a failure of innervation in the gastrointestinal tract [2]. ET_B receptor mutations are also associated with lethal white foal syndrome in horses as a result of limiting migration of melanocytes, pigment-producing cells found in hair follicles and skin.

[1] Guide to PHARMACOLOGY: ET_B receptor

[2] Davenport et al. (2016). Endothelin. Pharmacol Rev. 68:357-418. PMID: 26956245

[3] Shihoya et al. (2016). Activation mechanism of endothelin ETB receptor by endothelin-1. Nature, 537, 363-368. PMID: 27595334

Comments by Anthony Davenport

A team including the Gloriam Group at the University of Copenhagen (also the home of GPCRDB) have paper out in Nature Chemistry reporting the first total synthesis of YM-254890 and FR900359 [1] . These are related cyclic depsipeptide natural products that specifically and potently inhibit the Gq subfamily of G proteins, a relatively rare but useful and pharmacological property [3]. By a combination of solution and solid-phase approaches the team generated sufficient YM-254890 and FR900359 material for confirmation of the structures , pharmacological characterisation and the synthesis of ten new analogues of YM-254890 for SAR analysis. The paper also includes docking studies based on the X-ray crystal structure of YM-254890 in PDB 3AH8 [3]

[1] Xiong et al. (2016). Total synthesis and structure–activity relationship studies of a series of selective G protein inhibitors. Nat Chem, advance online publication, doi:10.1038/nchem.2577

[2] Schrage R, et.al. (2015) The experimental power of FR900359 to study Gq-regulated biological processes. Nat Commun. 14;6:10156. doi: 10.1038/ncomms10156, PMID 26658454

[3] Nishimura A. et. al.(2010) Structural basis for the specific inhibition of heterotrimeric Gq protein by a small molecule. Proc Natl Acad Sci; 107(31): 13666–13671. doi: 10.1073/pnas.1003553107, PMID 20639466

The two key potent ligands from the paper are included in the new GtoPdb release 2016.4. Details of this particular curation exercise are given in this blog post.
http://guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=9335

http://guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=9336

Comments by Chris Southan

Extracellular ATP is able to activate two families of cell-surface receptors, one of which is the ligand-gated ion channel family of P2X receptors. This family of cation channels is distinct from the remainder of the ligand-gated ion channels, as they are constructed of three (usually homomeric) subunits each with two transmembrane domains. Amongst the P2X receptors, the P2X3 is associated particularly with synaptic transmission in the sensory system and has, therefore, attracted a lot of attention as a potential target for novel analgesics and/or bladder dysfunction therapies.

In this report [1], multiple crystal structures of the P2X3 receptor are described, which allow a novel insight into the gating of a ligand-gated ion channel during the rest-agonist activated-refractory cycle, as well as with antagonist bound.

[1] Mansoor et al. (2016). X-ray structures define human P2X3 receptor gating cycle and antagonist action. Nature 538:66-71. doi: 10.1038/nature19367. [PMID 27626375].

Comments by Steve Alexander

In a recent article in Nature [1], Wu et al. present the cryo-electron microscopy structure of the rabbit Ca_v1.1 complex at a nominal resolution of 3.6 Å. Enrichment of purified channel particles without carbon film increased resolution and allowed to delineate structural features of the channel beyond those published by the authors in Science [2] six months earlier. The new structure reveals the channel in a (most likely) inactivated state (pore closed, voltage-sensors “up”), provides more complete structural detail of the α2δ-subunit and its interaction with extracellular surface of the pore-forming α1-subunit and unveils formation of a globular domain by direct interaction of the proximal C-terminal tail of α1 with its intracellular III-IV linker.
The new structural information provides new perspectives to address long-standing open questions. It will help to model human disease-related missense mutations within the Ca_v1.1 α1-subunit structure revealing the molecular mechanisms causing aberrant channel function, such as the formation of omega-pores in hypokalemic periodic paralysis [3]. The drug binding domains for Ca²⁺ channel blockers, widely used as antihypertensive drugs by blocking highly homologous Ca_v1.2 L-type channels in arterial resistance vessels, are highly conserved in Ca_v1.1. Together with recently published high resolution structure of the receptor sites for these drugs within the Ca²⁺-selective bacterial Na+-channel (NavAb) derivative Ca_vAb [4], the new Ca_v1.1 structure will now allow to further refine the molecular details of drug interactions with L-type Ca²⁺ channels. The unexpected finding of a globular domain formed by the proximal C-terminus and the cytoplasmic III-IV linker of the pore subunit could provide the structural missing link for understanding how the C-terminus mediates protein kinase A regulation of the channel and controls voltage- and Ca²⁺-dependent channel gating in Ca_v1.1 and other voltage-gated Ca²⁺ channels.
Finally, it will be interesting to see how well the Ca_v1.1 α1-subunit structure was predicted by homology modeling using bacterial Na⁺-channels (like Na_vAb) or mammalian K⁺-channels as a template [5].

[1] Wu et al. (2016). Structure of the voltage-gated Ca2+ channel Cav1.1 at 3.6 Å resolution. Nature 537:191-196. [PMID 27580036].
[2] Wu et al. (2015). Structure of the voltage-gated calcium channel Cav1.1 complex. Science 350: aad2395. [PMID 26680202].
[3] Wu et al. (2012). A calcium channel mutant mouse model of hypokalemic periodic paralysis. J. Clin. Invest. 122: 4580–4591. [PMID 23187123].
[4] Tang et al. (2016). Structural basis for inhibition of a voltage-gated Ca(2+) channel by Ca(2+) antagonist drugs. Nature 537: 117–121. [PMID 27556947].
[5] Tuluc et al. (2016). Molecular interactions in the voltage sensor controlling gating properties of Cav calcium channels. Structure 24:261–271. [PMID 26749449].

Comments by Jörg Striessnig (Department of Pharmacology and Toxicology – Institute of Pharmacy, Universität Innsbruck)

Changeux and Christopoulos have recently described in Cell [1] how common mechanisms link the allosteric sites of activation and response within the four major receptor families of ligand- and voltage-gated ion channels, G-protein-coupled receptors, nuclear hormone receptors, and receptor tyrosine kinases. As stated in the classical “Monod-Wyman-Changeux” model [2], the signal transduction mechanism operates through the selective stabilization of the particular state to which any ligand preferentially binds. Recent research shows that these states are affected by multiple factors including oligomerization, distinct conformational ensembles, intrinsically disordered regions, and allosteric modulatory sites. These processes can be perturbed by mutations that shift the equilibrium of receptor functional states and lead to disease [3]. Conversely, marketed medicines now include a large number of allosteric modulators with the advantages of fine-tune physiological responses and offer higher on-target selectivity via more diverse binding sites [4]. Such modulators can also display increased functional selectivity through biased agonism (i.e. the association with a distinct receptor conformation and signal routing). This review summarises the unifying mechanisms for the allosteric modulation of receptor classes and provides a clear demonstration of the associated pharmacological targeting opportunities.

[1] Changeux, J.-P. and A. Christopoulos (2016). Allosteric Modulation as a Unifying Mechanism for Receptor Function and Regulation. Cell. 166(5): p. 1084-1102. [PMID: 27565340]

[2] Monod, J., J. Wyman, and J.P. Changeux (1965). On the nature of allosteric transitions: a plausible model. J. Mol. Biol. 12: p.88-118. [PMID: 14343300]

[3] Changeux, J.-P. (2013). 50 years of allosteric interactions: the twists and turns of the models. Nat. Rev. Mol. Cell Biol. 14(12): p.819-29. [PMID: 24150612]

[4] Gentry, P.R., P.M. Sexton, and A. Christopoulos (2015). Novel Allosteric Modulators of G Protein-coupled Receptors. Journal of Biological Chemistry. 290(32): p.19478-19488. [PMID: 26100627]

Comments by David E. Gloriam (Department of Drug Design and Pharmacology, University of Copenhagen).

Lek et al. [1] in Nature, describes a tour de force large scale reference data set of high-quality protein-coding variation generated via the Exome Aggregation Consortium (ExAC) [2]. This covers 7,404,909  variants of different types that can be interrogated from an open browser set up by the team http://exac.broadinstitute.org/  [2]. Many interesting and important aspects of protein variation in both medical and evolutionary contexts are subject to statistical analysis and the results discussed. This includes loss-of function (LoF) with both clinical manifestations and consequent possible opportunities for pharmacological intervention. As just one example they investigate genetic intolerance to 179,774 high-confidence protein truncation variants (PTVd) that mapped to 3,230 highly LoF-intolerant genes. It turns out that 72% have no human disease phenotype in the OMIM or ClinVar databases. The Exac resource provides opportunities for detailed analysis of  functional variation as well as a filter for analysis of candidate pathogenic variants in Mendelian diseases. The paper also indicates that most of the proposed burden of Mendelian disease alleles per-person highlighted in previous reports, is due to misclassification in the literature and/or in databases [3]. In curating target records for GtoPdb the team have been finding it increasingly challenging to select between the many sources of protein variation and different levels of supporting evidence for the phenotypic consequences thereof. On the basis of these papers and our initial assessment of their database, we would now recommend Exac as a first-stop-shop for browsing the genomic variation landscape of GtoPdb targets, with GPCRdb, Swiss-Var, ClinVar and Orphanet as orthogonal backup.

[1] Lek et al. (2016). Analysis of protein-coding genetic variation in 60,706 humans. Nature 536, 285–291. [PMID: 27535533]

[2] Karczewski et al. (2016) The ExAC Browser: Displaying reference data information from over 60,000 exomes. bioRxiv (19 August 2016), 070581, doi:10.1101/070581 

[3] Walsh et al. (2016). Reassessment of Mendelian gene pathogenicity using 7,855 cardiomyopathy cases and 60,706 reference samples.
Genetics in Medicine. Aug 17. doi: 10.1038/gim.2016.90. [Epub ahead of print]. [PMID: 27532257]

Comments by Chris Southan

Manglik et al. [1] , writing in Nature, believe they may have found a new form of painkiller that works just as well as morphine but lacks its potentially lethal side effect. The authors have found it is not addictive by discovering a biased agonist that selectively targets the G-protein pathway over β-arrestin. Binding of agonists, such as morphine, to the μ-opioid-receptor cause very powerful reductions in the sensation of pain or analgesia via the G-protein signalling pathway but has the major side-effect of respiratory depression (the major cause of death in heroin addicts) and constipation. A further unwanted side effect limiting the use of morphine is addiction by activating the dopaminergic reward circuits. The authors show the new  μ-opioid agonist PZM21 selectively activates the G-protein signalling pathway to give the desired analgesia in animal models but does not activate β-arrestin pathway, so causes little respiratory depression or constipation nor alters the dopamine pathway so would be predicted not to be addictive.

The research is important as the authors report PZM21 in mice was comparable to morphine but longer lasting. Interestingly PZM21 reduced pain in the CNS but not spinal cord in mouse models.

A biased opioid agonist TRV130 is now in Phase III trials by the company Trevena Inc that is structurally unrelated to PZM21 but has a similar pharmacological profile. Taken together, the two compounds suggest that agonists biased to the G_i/o-pathway (rather than possible differences in other pharmacological properties such as pharmacokinetics)  represent a new strategy for pain control.

[1] Manglik A. et al. (2016). Structure-based discovery of opioid analgesics with reduced side effects. Nature. doi:10.1038/nature19112 advance online publication: 1-6.

Comments by Anthony Davenport

n.b. the  two relevant ligands curated into GtoPdb are show below. As a new entry 9286 PZM21 will go live in release 2106.4 (September) but TRV130 was already captured as ligand 7334

The key value of our curation is the extraction of chemistry-activity-target data from papers. Giving this relationship a formal structure in our database records not only provides direct value for users but this is also propagated globally by other databases that link to and/or subsume our content. Within the pharmacology/chemogenomic database ecosystem the largest  source of chemistry <> PubMed ID links is PubChem. Many PubChem records include depositor-provided cross-references to scientific articles in PubMed, both related to chemical structures and bioassay data. The recent paper by Kim and the PubChem team [1] includes a detailed statistical analysis of these relationships that add up to 5.6 million connections between 2.2 million PMIDs and 301,000 compound records (CIDs). The paper also describes and compares in detail the different depositors, publisher-supplied and Mesh chemisty <> PMID links.

Since we are one of the PubChem depositors of these relationships,  we were pleased to see not only a positive mention in this paper but also a detailed breakdown of our own contribution of 11,250 CID <> PMID relationships (presented in Table 1). Although these are small numbers compared to the total,  it should be noted that ~95% of these are generated automatically (i.e. not curated) by the IBM patent extraction system that they operated on PubMed in parallel with patent document processing up to 2010. Note this chemistry-to-literature connectivity is slowly being expanded by journals, include the British Journal of Pharmacology [2].

Comments by Curation Team