Database release 2018.2

Our second database release of the year, 2018.2, is now available. This update contains the following new features and content changes:

Content updates

GPCRs:
5-Hydroxytryptamine receptors
Adenosine receptors
Adrenoceptors
Histamine receptors
Opioid receptors
Lysophospholipid (S1P) receptors
Prostanoid receptors

NHRs:
Mineralocorticoid receptor
Peroxisome proliferator-activated receptors

Channels:
Transient Receptor Potential channels
Nav1.5

Enzymes:
Nitric oxide synthases
Cyclooxygenase
Phosphodiesterases, 3′,5′-cyclic nucleotide (PDEs)
Cyclin-dependent kinase (CDK) family
Mitogen-activated protein kinases (MAP kinases)
NADPH oxidases

Transporters:
Monoamine transporter subfamily

Others:
Heat shock proteins

New website features

Pharmacology Search Tool

In release 2018.1 we announced a new Pharmacology Search Tool allowing users to upload lists of target ids and find ligands to modulate them. We have now extended this tool to (optionally) search for other relevant ligands in ChEMBL v23. The ChEMBL data has been filtered according to the same rules we use for the ligand activity visualisation charts (see the help documentation for details) and as well as displaying the ChEMBL curated activity values, we also display their calculated -log pChEMBL value. An example of the results returned from this type of search is shown in Fig 1.

pharm_search_res

Figure 1. Example of results returned from a UniProt Accession search in the Pharmacology Search Tool, showing the top 3 GtoPdb and ChEMBL ligands. Results are ordered by total number of ligands in these databases that match search criteria.

New PDB ligand icon

As part of our increased emphasis on ligand structures (as seen with our synPHARM resource), we have introduced a new ligand icon for PDB entries. We display this on the ligand list and target interaction tables to indicate which ligands have PDB entries (orange circle with a white alpha helix across the centre), as shown in Fig 2.

PDB_icon_lig_list

Figure 2A. The ligand list showing the new PDB ligand icon in orange.

PDB_object_icon

Figure 2B. A target inhibitor table showing the new PDB ligand icon in orange.

Sponsored Tocris product links

We have collaborated with Tocris as a quality supplier of many of the ligands in GtoPdb by adding links from our ligand pages out to the matching Tocris products. An example, which can be found on the ligand page beneath the summary tab, is shown in Fig 3. In total there are links to 1198 Tocris products.

tocris_link

Figure 3. Ligand summary page showing a link to the Tocris product.

Other updates

BJP/BJCP linking

As an adjunct to our successful entity-linking initiative for the BJP and more recently BJCP, we have instigated a process whereby, on manuscript acceptance and their own marking-up of GtoPdb links, authors alert us directly to key entities from their studies that are not in our database. In most cases, we then add the missing ligands. This has the advantages for both the author and the journal of not only adding their reference into GtoPdb but also the paper gains PubChem PubMed reciprocal linking derived from our PubChem ligand submissions. Examples from this release include GS-458967 from BJP and esaxerenone from BJCP.

BACE1 in doubt as an Alzheimer’s drug target

The target entry for the Alzheimer’s drug target BACE1 underwent key updates. The first of these was to add a new reference for the full report published just last week on the Phase III failure of the lead Merck BACE1 inhibitor verubecestat. Unfortunately, this paper now casts doubt on the target validation status and thus the future for this entire class of compounds pursued intensively for over 18 years. Notwithstanding, several ligands on the BACE1 list may yet complete their clinical evaluation (these will be joined by the latest  development candidate from Pfizer as PF-06751979 in 2018.3)

SynPHARM article

We are pleased to report that an open pre-print version (i.e. pending changes compared to the eventually accepted journal version) of our manuscript describing our SynPharm resource is now on-line.

How-to-Guide 

We are also pleased to report the publication of  Accessing Expert‐Curated Pharmacological Data in the IUPHAR/BPS Guide to PHARMACOLOGY. It is not indexed in PubMed yet but note that the PDF is free to access until the end of May (so pull it down while you can! – but we could send you one if you miss the window).  It includes useful examples of how to use both GtoPdb and GtoImmuPdb as a supplement to our online help and FAQ.

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Hot topic: Cryo-EM structure of the adenosine A2A receptor coupled to an engineered heterotrimeric G protein

The A2A adenosine receptor is densely expressed in dopamine-rich areas of the brain and in the vasculature. It is the target of an adjunct medication for Parkinson’s Disease, istradefylline in Japan, an A2A receptor antagonist.

The A2A adenosine receptor is an example of a Gs-coupled receptor, activation of which in the cardiovascular system leads to inhibition of platelet aggregation and vasorelaxation. This new report (1) highlights the link between the receptor and the G protein to focus on areas of unexpected flexibility in the ligand binding region. Further, classical understanding of receptor:G protein interaction identifies a prominent role for the third intracellular loop and the proximal end of the C-terminus (in some GPCR, such as the beta2-AR, a fourth intracellular loop is formed by palmitoylation of an intracellular cysteine residue, which the A2A lacks). The model generated from this cryo-EM study with a nanobody suggests a potentially novel role for an interaction between the first intracellular loop and the Gbeta subunit.

Comments by Steve Alexander (@mqzspa)

(1) Garcia-Nafría J et al. (2018). Cryo-EM structure of the adenosine A2A receptor coupled to an engineered heterotrimeric G protein. eLife, 7. pii: e35946. doi: 10.7554/eLife.35946. [PMID: 29726815]

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Hot topic: Conformational plasticity in the selectivity filter of the TRPV2 ion channel

The TRPV2 ion channel is the less well-characterised relative of the TRPV1 or vanilloid receptor that is activated by capsaicin. TRPV2 channels have many similarities to the TRPV1 channels, in that they are homotetrameric and respond to some of the same ligands (natural products such as cannabinoids) as well as being triggered at elevated temperatures. This study (1) focusses on a different common feature of the whole Transient Receptor Potential family, which are often described as non-selective cation channels. Using comparative analysis of crystals structures in which calcium is bound with and without an agonist, resiniferatoxin, present. The authors suggest that this agonist evokes a symmetrical opening of a selectivity filter gate, which permits increased permeation of calcium ions and also larger organic cations, such as the dye Yo-PRO-1.

Comments by Steve Alexander (@mqzspa)

(1) Zubcevic L et al. (2018). Conformational plasticity in the selectivity filter of the TRPV2 ion channel. Nat Struc Mol Biol., 25:405-415. doi:10.1038/s41594-018-0059-z. [Abstract]

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Hot topic: 3D structure of the P2X3 receptor bound to a negative allosteric modifier, identifies a binding site that is a target for development of novel therapeutic agents

Negative allosteric modulators (NAMs) are of great interest in drug development because they offer improved scope for the production of receptor antagonists with enhanced subtype-selectivity. Indeed, many NAMs are already on the market or undergoing clinical trials. NAMs act by binding to sites within receptors that are distinct from the primary, orthosteric ligand binding site and can inhibit the structural rearrangements of a receptor that are induced by orthosteric agonist binding.

P2X receptors are ligand-gated cation channels for which ATP is the endogenous orthosteric agonist. They are expressed throughout the body and the evidence indicates that they have numerous functions, including in sympathetic and parasympathetic neurotransmission, perception of sound, taste and pain, and immune regulation. Seven P2X subunits have been identified, which form trimers, to produce at least twelve different receptor subtypes. A major issue within the field has been a lack of selective antagonists for most P2X subtypes. This is unsurprising given the amino acid sequence similarity within the ATP binding site. Several selective NAMs have now been developed, but little is known about where in receptors they act and how exactly they inhibit receptor activation.

AF-219 is small molecule NAM at P2X3 receptors that was reported to be effective in a phase II clinical trial for treatment of refractory chronic cough. Wang et al., (1) combined X-ray crystallography, molecular modelling, and mutagenesis, to identify the site and mode of action of AF-219. P2X3 receptors are composed of three subunits, each of which adopts a conformation that could be likened to the shape of a leaping dolphin. The tail represents the transmembrane-spanning regions, the upper body the bulk of the extracellular loop and the head the most distal part of the extracellular loop. Also attached to the body are three structurally-distinct elements: the dorsal fin, the right flipper, and the left flipper. As a trimer, the subunits wrap round each other to produce a structure that resembles a chalice.

The AF-219 binding site is formed by the lower body and dorsal fin of one subunit and the lower body and left flipper of an adjacent subunit. Mutational analysis identified which amino acid residues within this pocket are essential for AF-219 binding, whilst in silico modelling showed that the small molecule P2X3 NAMS, AF-353, RO-51, RO-3 and TCP 262, but not the large NAMS suramin and PPADS, also bind to the same site. Activation of P2X3 receptors by ATP closes the binding cavity, so by occupying it, AF-219 prevents the protein structural rearrangements that lead to opening of the P2X3 receptor ion pore.

This identification of the AF-219 NAM binding site in P2X3 receptors is an opportunity for rational, intelligent drug design. It enables virtual screening of compound libraries, with the aim of identifying potential new molecular core structures, which can then be modified in order to optimise the structure of a novel NAM. In addition, this site differs among P2X receptor subtypes, so it is highly possible that drugs with greatly enhanced subtype-selectivity can be developed.

Comments by Dr. Charles Kennedy, University of Strathclyde

(1) Wang J, Wang Y, Cui WW, Huang Y, Yang Y, Liu Y, Zhao WS, Cheng XY, Sun WS, Cao P, Zhu MX, Wang R, Hattori M, Yu Y. (2018). Druggable negative allosteric site of P2X3 receptors. Proc Natl Acad Sci U S A. 2018 pii: 201800907. doi: 10.1073/pnas.1800907115. [PMID:  29674445]

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Hot topic: 3D structures of the closed acid-sensing ion channel (ASIC) shed light on the activation mechanism of these neuronal ion channels

ASICs are potential drug targets of interest. Their activation mechanism has however remained elusive. ASICs are neuronal, proton-gated, sodium-permeable channels that are expressed in the central and peripheral nervous system of vertebrates. They form a subfamily of the Epithelial Na channel / degenerin channel family, and contribute to pain sensation, fear, learning, and neurodegeneration after ischemic stroke. Depending on the extracellular pH, they exist in either one of three functional states: closed (resting), open and desensitized. While ASICs are at physiological pH 7.4 in the closed state, they open briefly upon extracellular acidification, before entering the non-conducting desensitized state. Crystal structures of the chicken ASIC1 channel in the desensitized and the open state were published several years ago. This structural information allowed, together with observations from functional studies, an understanding of the transitions between the open and the desensitized state. In contrast, the absence of structural information on the closed conformation of ASICs precluded so far a molecular understanding of their activation mechanism.

The Gouaux laboratory has now published structures of the homotrimeric chicken ASIC1 obtained at high pH by X-ray crystallography (2.95 Å resolution) and by single particle cryo-electron microscopy (3.7 Å) (1). These structures show a channel with a closed pore, representing likely the closed state. The overall structural organization is the same in all ASIC 3D structures published so far: each subunit consists of a large, complex ectodomain, two transmembrane domains, and short N- and C-termini (whose structure has not been resolved yet). The channel is formed by three identical subunits that are arranged around the central ion pore. A vestibule containing many acidic residues, the “acidic pocket”, is located on the outward-facing side of the ectodomain of each subunit, at 40-50 Å from the membrane. The main difference in the ectodomain between the closed ASIC structures and previously published open and desensitized structures is a wide opening of the acidic pocket in the structure of the closed channel.

Based on the comparison of closed, open and desensitized structures, the authors suggest the following activation mechanism: At physiological pH 7.4 the channel pore is closed and the acidic pocket has adapted an extended conformation. Extracellular acidification protonates acidic residues of the acidic pocket, thereby reducing repulsion between such residues and leading to a collapse of the acidic pocket. This movement is transmitted via central channel domains to the transmembrane helices, and leads to opening of the channel pore. A short time later, an additional movement in the central domains uncouples the ion pore from the acidic pocket and allows the transmembrane domains to relax to the non-conducting desensitized conformation. The acidic pocket will adapt its extended conformation only once the extracellular pH has returned to higher values.

This new 3D structure is undoubtedly a breakthrough in the understanding of the molecular mechanisms of ASIC activity. Some open questions remain however:

Several studies have shown that protonation events in domains other than the acidic pocket contribute to activation and desensitization, and it has also been shown that a channel in which most of the acidic residues in the acidic pocket have been neutralized can still be opened by extracellular acidification. These studies suggest that an important part of the drive for the conformational changes comes from protonation events outside the acidic pocket. This is different from the activation mechanism proposed by Yoder and colleagues, which relies on protonation events in the acidic pocket.

The cytoplasmic N- and C-termini of ASIC subunits contain sites important for ASIC function and ion selectivity. So far there is no structural information on these intracellular parts available. Future cryo-electron microscopy approaches will hopefully have the power to resolve the conformation of these domains.

Comments by Stephan Kellenberger, Université de Lausanne, Switzerland

1. Yoder, N., Yoshioka, C., and Gouaux, E. (2018) Gating mechanisms of acid-sensing ion channels. Nature, 555: 397-401. doi: 10.1038/nature25782. [PMID:29513651]

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Hot topic: Engineered mini G proteins provide a useful tool for studying the activation of GPCRs in living cells

In order to stabilize the GPCR-G protein complex, an agonist must be bound to the receptor and the alpha subunit of the heterotrimer must be in a nucleotide-free state. Ground-breaking work by expert crystallographers made use of so-called mini G (mG) proteins to stabilize the active conformation of the adenosine A2A receptor in the presence of agonist and guanine nucleotides, but in the absence of Gβγ [1]. These engineered G proteins behave in a way that mimics the nucleotide-free state despite being bound to GDP; thus, they can be seen as conformational sensors of the active receptor state. This work paved the way for another study recently published in the Journal of Biological Chemistry led by Nevin A. Lambert that looked to build on this minimalistic approach to see if representative mG proteins from the four subclasses (Gs, Gi/o, Gq/11 and G12/13) could 1) detect active GPCRs and 2) retain coupling specificity [2]. Using bioluminescence resonance energy transfer (BRET) assays, the interaction between mGs, mGsi, mGsq or mG12 with prototypical GPCRs was quantified to examine whether these tools could reveal ligand efficacy/potency and G protein specificity. This was not only confirmed through exhaustive validation, but surprisingly uncovered secondary coupling interactions that might be of potential interest for follow-up studies. The GPCR superfamily comprises more than 800 GPCRs – most of which we know very little about. These elegant tools should prove valuable in increasing our knowledge about the lesser known GPCRs as well as allow for the discovery of G protein subtype-biased ligands and for unravelling receptor coupling complexity.

Comments by Shane C. Wright and Gunnar Schulte, Karolinska Institute

References

  1. Carpenter B, Nehme R, Warne T, Leslie AG and Tate CG. (2016) Structure of the adenosine A(2A) receptor bound to an engineered G protein. Nature, 536 (7614): 104-107. [PMID:27462812]
  2. Wan Q, Okashah N, Inoue A, Nehme R, Carpenter B, Tate CG and Lambert NA. (2018) Mini G protein probes for active G protein-coupled receptors (GPCRs) in live cells. J Biol Chem. pii: jbc.RA118.001975. doi: 10.1074/jbc.RA118.001975. [Epub ahead of print] [PMID:29523687]
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Hot Topic: Unexplored therapeutic opportunities in the human genome

Contemporary drug discovery is dominated by two related  themes. The first of these is target validation upon which the sustainability of pharmaceutical R&D (in both the commercial and academic sectors) crucially depends.  The second is the size of the pool of human proteins that are/could become tractable to being progressed towards clinical efficacy as their final validation step (otherwise known as the druggable proteome).  This usefully detailed review, by a large team of authors, touches on both themes but with a focus on how the community might increase the target pool by data-driven knowledge expansion for hitherto less well characterised proteins [1].

As explained in the paper, this shortfall is being addressed by the NIH Illuminating the Druggable Genome (IDG) project since 2014 [2].  As essential reading for those engaging with the  intersects between pharmacology and drug discovery, just a few aspects can be picked out. One of these is their formalisation of a target development level (TDL) classification scheme of Tclin (clinical evidence), Tchem (chemical modulators), Tbio (biological data)  and Tdark related to the depth of investigation.  This “dark” category encompasses proteins with the least current knowledge (i.e. unvalidated potential targets) and a low number of (if  any) molecular probes.  Included in this are of course the orphan GPCRs that have been the subject of previous Hot Topics in their own right [3].

The authors not only point to many additional resources but also present a wealth of detailed statistics on many aspects of drug targets.  These included (Table 1) that eight olfactory receptors have Tbio level data.  Another nugget was the fact that phenomenological responses following radiation therapy is a bona fide biological functional characterisation approach that few of us are aware of. Last but not least, we were pleased to see GtoPdb [4] cited as one of the sources included in this impressive analysis.

For the record, our own curatorially-supported human druggable target list encompasses 1496 proteins with quantitative ligand interactions.  This can be found via the UniProt cross-reference. (n.b. this number will change slightly as the links from our own latest database release will update in the forthcoming UniProt release).

Comments by Chris Southan, IUPHAR/BPS Guide to PHARMACOLOGY, @cdsouthan
References
  1. Oprea TI et al. (2018). Unexplored therapeutic opportunities in the human genome. Nat Rev Drug Discov. doi: 10.1038/nrd.2018.14 [Epub ahead of print] [PMID:29472638]
  2. Illuminating the Druggable Genome (IDG) Program. https://commonfund.nih.gov/idg
  3. Hot topic: The G Protein-Coupled Receptors deorphanization landscape.  https://blog.guidetopharmacology.org/2018/02/28/hot-topic-the-g-protein-coupled-receptors-deorphanization-landscape/
  4. Southan C et al. (2016) The IUPHAR/BPS Guide to PHARMACOLOGY in 2016: towards curated quantitative interactions between 1300 protein targets and 6000 ligands.  Nucleic Acids Res. 44(D1):D1054-68. doi: 10.1093/nar/gkv1037. [PMID:26464438]

 

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