Hot Topics: New Cannabinoid Receptors Structures

Cannabinoid receptors respond to multiple endogenous fatty acid derivatives and are often divided into neuronal-associated CB1 receptors and immune cell-associated CB2 receptors. Both receptors are GPCR, coupled predominantly to Gi, and have cytoprotective properties. The predominant psychotropic agent in Cannabis, THC, acts as a partial agonist at both receptors. CB1 patho/physiological responses are often characterised as analgesic, rewarding, orexigenic, hypothermic and amnestic, while CB2 receptors are mostly associated with anti-inflammatory effects.

In many countries, synthetic cannabinoids have become a social issue, with a prevalence of use amongst the homeless and incarcerated, with even a number of deaths attributed to these agents. Although all the molecular mechanisms of action of these synthetic cannabinoids are yet to be defined, one feature they have in common is a high potency and high efficacy profile at CB1 receptors. Kumar and colleagues [1] report a CB1 receptor:Gi complex, where the receptor is bound to a synthetic cannabinoid, MDMB-FUBINACA. The authors report an agonist binding-evoked conformational switch involving residues in TM3 and TM6, which they suggest underlies the high affinity of this synthetic cannabinoid. Furthermore, they conduct in silico simulations to suggest a lateral path of entry for the synthetic cannabinoid between TM1 and TM7 rather than the ‘traditional’ extracellular point of ingress. This lateral diffusion model has been suggested for a number of lipid-binding GPCR.

There is a second cannabinoid receptor crystal structure in the same journal, which focusses on the CB2 receptor [2]. Based on the primary sequences of the two human receptors, there is limited structural identity between CB1 and CB2 (~40 %), although the overlap is much higher in the transmembrane domains, as might be expected, given they bind a number of structurally-diverse ligands with little discrimination (e.g. CP55940, WIN55212-2 and HU210). Li et al report the first crystal structure of the CB2 receptor. In this version, a novel high affinity antagonist/inverse agonist AM10257 was bound to the receptor for crystallisation. The resultant structure shows a number of similarities with the antagonist-bound structure of the CB1 receptor, although notably the extracellular portions of the two receptors diverged markedly. Slightly surprisingly, a close resemblance to the agonist-bound CB1 receptor was identified, which lead them to investigate CB1 receptor function of the novel CB2 antagonist, which turned out to be a low efficacy CB1 receptor agonist.

Comments by Steve Alexander (@mqzspa)

[1] Kumar KK et al. (2018). Structure of a Signaling Cannabinoid Receptor 1-G Protein Complex. Cell, pii: S0092-8674(18)31565-4. doi: 10.1016/j.cell.2018.11.040. [Epub ahead of print]. [PMID:30639101].

[2] Li X et al. (2018). Crystal Structure of the Human Cannabinoid Receptor CB2. Cell, pii: S0092-8674(18)31625-8. doi: 10.1016/j.cell.2018.12.011. [Epub ahead of print]. [PMID:30639103].

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GtoPdb at BPS Pharmacology 2018

The IUPHAR/BPS Guide to Pharmacology was represented at the recent BPS Pharmacology 2018 meeting (London, UK, 18-20 Dec 2018).

Tuesday 18th Dec

On Tuesday we had two significant presentations. Firstly, a late-breaking poster on the IUPHAR/MMV Guide to Malaria Pharmacology. This is an under-development extension to the database to curate in anti-malarial ligands and Plasmodium targets for approved drugs.

Poster: Introducing a new resource: the capture of drugs, leads and targets in the IUPHAR/MMV Guide to MALARIA PHARMACOLOGY (Presented by Dr. Chris Southan & Dr. Simon D. Harding)

Secondly, Dr. Southan presented on the challenges and tribulations of curating peptides in the Guide to Pharmacology. His slides are available below.

Oral Presentation: Tribulations of curating published key bioactive peptides for the Guide to PHARMACOLOGY

Thursday 20th Dec

On the Thursday by three more presentations. Firstly, Dr. Harding presented a flash presentation and poster on new features and updates to the Guide to Pharmacology in 2018, which was awarded the daily flash poster prize.

Poster: The IUPHAR/BPS Guide to PHARMACOLOGY in 2018: new features and updates

Also during the poster session Dr. Southan presented his second poster looking at how we can identify the most pharmacologically significant proteins.

Poster: Will the real pharmacologically significant proteins please stand up?

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Hot Topic: GPR37/GPR37L1 and the putative pairing with prosaptide/PSAP

Comments by Dr. Nicola J. Smith, National Heart Foundation Future Leader Fellow & Group Leader, Molecular Pharmacology Laboratory, Victor Chang Cardiac Research Institute, Australia

As is often the case with orphan GPCRs, assigning the endogenous ligand has been controversial for the closely related peptide family orphans, GPR37 and GPR37L1. In 2013, Randy Hall and his team (PubMed: 23690594) first reported an association between both centrally-expressed orphan GPCRs and prosaposin (PSAP) and prosaptide (TX14A), the synthetic active epitope of PSAP. Since that time there has been much debate in the field about whether this pairing is correct, with some authors corroborating the findings (PubMed: 24371137; 30010619, 28795439) and others not (PubMed: 23396314; 27072655; 28688853). Note that Head Activator, found in Hydra, was earlier reported as a ligand (PubMed: 16443751) but was quickly discredited (PubMed:28688853; 23686350).

A recent paper by Sergey Kasparov’s laboratory in Bristol has added further fuel to the fire. In a series of well controlled experiments, Liu et al. (PubMed: 30260505) provided convincing evidence that prosaptide is cyto- and neuro-protective and promotes chemotaxis. They are also the first group to demonstrate an effect of prosaptide at a more physiologically plausible potency. At the same time, Bang et al. (PubMed: 30010619) published a ground-breaking paper linking GPR37 expression to macrophage function. Moreover, they proposed a second, more potent ligand for GPR37 (GPR37L1 was not studied): the pro-resolving mediator neuroprotectin D1 (NPD1). Using HEK293 cells expressing GPR37, NPD1 was a potent stimulator of Gαi/o-dependent calcium flux; findings that were corroborated in macrophages isolated from wild type, but not GPR37 knock-out, mice (PubMed: 30010619). Thus, it may be that the endogenous ligand for GPR37 (and perhaps GPR37L1?) is not a peptide after all, but a lipid.

These two studies, while exciting, do little to help us resolve the conundrum that is PSAP/prosaptide and GPR37/GPR37L1. At the very least, it seems likely that prosaptide, if not the highest affinity endogenous ligand at GPR37, is certainly capable of signalling through GPR37 to stimulate Gαi/o signal transduction (whether it is the most potent endogenous agonist will be shown in time as independent groups seek to validate the actions of NPD1).

But what of GPR37L1? This is harder to answer as a number of studies linking GPR37L1 to PSAP/prosaptide have been performed in double GPR37/GPR37L1 knock-out backgrounds or inappropriate tissue models. For example, in the original paper connecting prosaptide to the receptors, the authors claimed prosaptide acted through both GPR37 and GPR37L1 in primary astrocytes, despite the fact that their Western blots demonstrated marked GPR37 expression in comparison to GPR37L1 in the cells (PubMed: 23690594). More recently, they failed to recapitulate this initial pairing in a HEK293 model (PubMed: 28688853). The absence of GPR37L1 in primary astrocytes is consistent with the animal knock-out work of Marazziti et al. (PubMed: 24062445), who showed that GPR37L1 protein was barely detectable before post-natal day 15, which is after the window for isolating primary astrocyte cultures (confirmed by PubMed: 28795439). Coleman et al. (PubMed: 27072655) overcame this expression issue, with difficulty, by using cerebellar slice cultures in vitro to examine Gαs, but not prosaptide, signalling in wild type and knock-out tissue.

In the neuroprotection paper by Liu et al. (PubMed: 30260505) it is clear that prosaptide or PSAP are exhibiting an effect on the cells. By depleting astrocytes of PSAP and then reintroducing prosaptide exogenously, there is an obvious effect on cell migration, cytotoxicity and neuroprotection – phenotypes that are all lost when shRNA knocking down expression of both GPR37 and GPR37L1 are used. Frustratingly, though, the use of a double knock-down approach makes it impossible to ascribe a specific effect to GPR37L1. While GPR37 and GPR37L1 are very closely related by phylogeny and have highly similsar binding sites (PubMed: 27992882), this does not mean that prosaptide is a ligand at both receptors, nor that both receptors signal via the same G proteins (another area of controversy for GPR37L1, where contradictory studies including two by the same team show either Gαi/o or Gαs signaling: PubMed: 23690594, 30260505 vs 27072655, 28688853). Thus, the failure to use single receptor knock-out/knock-downs, or isolate cells with endogenous expression of GPR37L1, represent major limitations in these studies.

Other than the confounding effects of both GPR37 and GPR37L1 deletion in tested cells, what are other reasons that could explain the inconsistencies between studies? Kasparov and colleagues (PubMed: 30260505) attribute this to cellular background, stating that previous studies that failed to confirm prosaptide/GPR37L1 coupling (PubMed: 27072655, 28688853) used HEK293 cells that must be lacking in the necessary endogenous machinery for signal transduction (PubMed: 30260505). To support this claim, they turned to the PRESTO-Tango assay in HEK293 cells to demonstrate prosaptide stimulation could not lead to GPR37L1-dependent recruitment of beta-arrestin. The assay choice is surprising because previous beta-arrestin-based screens at GPR37L1 have failed to show that the receptor can indeed recruit arrestins (PubMed: 23396314, 25895059), and Liu et al. (PubMed: 30260505) did not provide evidence that recruitment was intact in a more physiologically relevant cellular background. Most puzzling though is that the original paper that identified prosaptide and PSAP as GPR37/37L1 ligands used HEK293 cells to make the original pairing (PubMed: 23690594). They also refute the physiological relevance of high constitutive Gαs signalling reported by Coleman et al., even though Coleman et al. demonstrated higher cAMP accumulation in cerebellar brain slices from wild type mice when compared to GPR37L1-/- (PubMed: 27072655).

Further muddying the waters, the physiological role of GPR37L1 itself remains enigmatic. For example, Min et al. (PubMed: 20100464) initially reported GPR37L1 null mice to have a staggering 62 mmHg increase in systolic blood pressure when compared to a cardiac-specific overexpressing model, with the presence of concomitant left ventricular hypertrophy. However, Coleman et al. (PubMed: 29625592) found a far more marginal cardiovascular phenotype, with a small increase in blood pressure evident in female mice only. Notably, male GPR37L1 knock-out mice appeared to be more susceptible to cardiovascular stressors, while females were cardioprotected (PubMed: 29625592). In terms of a developmental phenotype, Marazziti et al. (PubMed: 24062445) found that GPR37L1 null mice displayed precocious cerebellar development with enhanced performance in a rotarod test up to 1 year of age. More recently, though, Jolly et al. (PubMed: 28795439) failed to confirm a behavioural difference in their own GPR37L1 knock out mice. The links between GPR37L1 and neurological defects are also confounded by the fact that GPR37 also needs to be deleted in mice for a clear phenotype to be evident. For example, while a single point mutation in GPR37L1 (K349N) in a highly consanguineous family appeared to be causative of fatal progressive myoclonus epilepsy, the mouse phenotype was most pronounced in double GPR37/GPR37L1 knock-out animals (PubMed: 28688853). In vitro studies of the GPR37L1 K349N mutant found no difference between it and the wild type receptor in terms of receptor expression, processing, signalling or ubiquitination (PubMed: 28688853). In the absence of a transgenic K349N mutant mouse, or any confirmed synthetic agonists or antagonists, the authors then assessed seizure susceptibility in knock-out mice of either GPR37L1, GPR37 or both receptors. Interestingly, using the 6Hz-induced seizure model, the GPR37-/- mice appeared to have a more pronounced phenotype than the GPR37L1-/- mice, while double KO mice were extremely susceptible to seizures at all frequencies tested. GPR37 and double KO mice both displayed more spontaneous seizures, although curiously in the flurothyl-induced seizure model only GPR37L1-/- differed from wild type. Thus, conclusive links between GPR37L1 and a specific physiological or pathophysiological state remain to be provided and it seems in general that we are far from understanding the true biology and pharmacology of the receptor.

 

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Hot Topic: Somatic APP gene recombination in normal and Alzheimer’s disease neurons

A new facet of the human brain has been reported [1] involving a first example of somatic gene recombination in neurons, representing a normal neural mechanism whose disruption could underlie the most common (sporadic) forms of Alzheimer’s disease. Mosaic and somatic recombination of Amyloid Precursor Protein (APP) was identified in this well-known Alzheimer’s disease gene, where increased copies and mutations in rare families or Down syndrome are considered causal. Recombination generates thousands of previously unknown gene variants characterized as “genomic complementary DNAs” or “gencDNAs” that could show identical sequences to cDNAs copied from brain-specific spliced RNAs, as well as myriad truncated forms characterized by exonic deletions and “intraexonic junctions” to produce novel sequences that become “retro-inserted” into the genome of single neurons, with neurons showing from 0 to 13 copies. Recombination appeared to require gene transcription, reverse transcriptase activity and DNA strand breaks. Both forms and numbers of APP gencDNAs were altered and increased in sporadic Alzheimer’s disease. Recombination might normally provide a way to alter post-mitotic neuronal genomes to “record” preferred gene variants for later “playback” without a need for gene splicing, towards optimizing or fine-tuning gene expression, representing a form of memory. The involvement of reverse transcriptase activities implicate potential Alzheimer’s disease therapeutics using reverse transcriptase inhibitors, a possibility supported epidemiologically by relatively rare cases of Alzheimer’s disease in aged HIV patients. Recombination could generate new therapeutic targets. Other recombined genes and affected diseases are possible.

Comments by Jerold Chun, Sanford Burnham Prebys Medical Discovery Institute

(1) Lee MH et al. (2018). Somatic APP gene recombination in Alzheimer’s disease and normal neurons. Nature, doi: 10.1038/s41586-018-0718-6. [Epub ahead of print]. [PMID:30464338].

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Hot Topic: Cellular thermal shift assays to measure ligand-to-target engagement

The cellular thermal shift assay (CETSA) was introduced in July of 2013 as a means to investigate drug target engagement inside live cells and tissues (1). The underlying principle of CETSA is simple – it relies on the thermostability of each investigated protein and how this is altered by ligand binding. Experimentally these changes are assessed by applying a transient heat-pulse to the samples. This results in rapid rearrangements of established equilibria such that proteins denature and aggregate unless stabilised by ligand (1,2). The simplicity of CETSA has allowed prompt adoption in the literature but the importance of rapid changes in ligand binding is still not well recognised.

To explore these considerations we systematically varied both the heat-pulse temperature and duration in CETSA using p38a as our model system (3). Studies spanning seven different heating times and over a 13°C temperature interval show apparent potency changes over four orders of magnitude. These studies demonstrate how quantitative comparisons with functional cellular data require an understanding of the temperature dependence of the interactions under study. Our publications also discuss critical technology developments that allow shorter heating times.  These can now be down in the 10s of seconds range to minimize ligand rearrangements and heat-induced changes to cell permeability.

Comments by Dr. Thomas Lundbäck,  Associate Director, Mechanistic Biology & Profiling, Discovery Sciences, AstraZeneca R&D, Gothenburg,  Sweden,  thomas.lundback@astrazeneca.com

  1. Martinez Molina, D.; Jafari, R.; Ignatushchenko, M.; Seki, T.; Larsson, E. A.; Dan, C.; Sreekumar, L.; Cao, Y.; Nordlund, P., Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science 2013, 341 (6141), 84-7 (PMID 23828940).
  2.  Jafari, R.; Almqvist, H.; Axelsson, H.; Ignatushchenko, M.; Lundbäck, T.; Nordlund, P.; Martinez Molina, D., The cellular thermal shift assay for evaluating drug-target interactions in cells. Nat Protoc 2014, 9 (9), 2100-22 (PMID 25101824).
  3.  Seashore-Ludlow, B.; Axelsson, H.; Almqvist, H.; Dahlgren, B.; Jonsson, M.; Lundbäck, T., Quantitative Interpretation of Intracellular Drug Binding and Kinetics Using the Cellular Thermal Shift Assay. Biochemistry Nov 2018 (PMID 30418016).
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Immunopharmacology: challenges, opportunities and research tools. Edinburgh 1st-2nd October 2018.

At the beginning of October 2018 we held a meeting in Edinburgh focussed on the launch of the IUPHAR Guide to IMMUNOPHARMACOLOGY. Invited speakers contributed to productive discussions on the varying challenges and opportunities in immunopharmacology research.

Immunopharmacology: The New Frontier

There has been immense progress in immunopharmacology, but there are insufficient links between the immunological and pharmacological sciences. Thus, we have set up several initiatives.

  • IUPHAR set up an immunopharmacology section (Immuphar) chaired by Francesca Levi-Schaffer.

  • IUPHAR has signed an agreement with International Union of Immunological Sciences (IUIS, President Alberto Mantovani, who has also made major contributions to the field of check-point inhibitors) to ensure collaboration and cooperation.

  • IUPHAR, NC-IUPHAR (chair Steve Alexander), the University of Edinburgh (PI, Pr Jamie Davies) and the Edinburgh database group (IUPHAR/BPS Guide to Pharmacology; www.guidetopharmacology.org) have been able to set up a new database on the drug targets in immunopharmacology, financed by a major grant from the Wellcome Trust. This is www.guidetoimmunopharmacology.org, which has been recently launched and is freely available to all. The BPS finance two staff in the Edinburgh group for which IUPHAR is immensely grateful.

  • To celebrate this launch, a focussed immunopharmacology meeting was organised, which included the Anthony Harmar memorial lecture. This report provides a a summary of the meeting presentations, discussions and outcomes.

The launch of GtoImmuPdb has also been reported in a Nature Review Immunology Web Watch article: A new guide to immunopharmacology (https://doi.org/10.1038/s41577-018-0079-2)

Please read our detail meeting report which summarises the presentations, discussions and outcomes. Download the Meeting Report (PDF)

Access slidesets from the presentations below (or on our website):

Meeting Presentations

 

Anthony Harmer Memorial Lecture: Decision-making in lung immunity Prof. Tracy Hussell Download slides: pptx | pdf
The Guide to IMMUNOPHARMACOLOGY Dr. Elena Faccenda, Dr. Chris Southan and Dr. Simon Harding Download slides: pptx | pdf
Macrophage plasticity in immunopathology and cancer: from bench to bedside Prof. Alberto Mantovani Download slides: pptx | pdf
Targeting Pattern Recognition Receptor signalling for therapeutic approaches Prof. Clare Bryant slides not available
Discovering the right target in inflammatory disease Prof. Iain McInnes slides not available
Is Atherosclerosis a Systemic or a Vascular Immune Disease? Prof. Pasquale Maffia slides not available
Inhibit Activation or Activate Inhibition of Mast Cells and Eosinophils: Which Weapon is Better to Fight Allergic Diseases? Prof. Francesca Levi-Schaffer Download slides: pptx | pdf
IUPHAR: natural products and immunology Prof. Michael Spedding Download slides: pptx | pdf
Human type I interferon up regulation – worth targeting? Prof. Yanick Crow Download slides: pptx | pdf
A review on kinase targets in immunological indications Dr. Dorian Fabbro Download slides: pdf
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The Anthony Harmar Memorial Lecture 2018: Prof. Tracy Hussell

Prof. Tracy Hussell

Lung Disease: think about disease in a different way

Hussell Tracey (Altered)Tracy Hussell is Director, Manchester Collaborative Centre for Inflammation Research (MCCIR) and Professor of Inflammatory Disease, University of Manchester, Oxford Road, Manchester, M13 9PT.

To be healthy is an active process so health must be continually maintained. Is disease a process that can’t sense it is healthy again? We all have different set points. Sterile inflammation can be created by a single missing factor. Generally, while the lung epithelium is intact, then the tissue is non-activated. Epithelial damage is a critical driver, which may permanently change macrophages and the basal state in the lung. Airways macrophages are critical – when washed out, tissues change very quickly.

CD200R transmits negative signal to macrophages and ligand is on epithelium cells. Then antigen relieves signal. Axl continually recognises gas 6 on apoptosis so don’t activate with apoptosis.

Resolution of inflammation gives a different macrophage population – twice as many as before inflammation, which may survey the environment, resolving from a severe inflammatory event, to a new state. There are therapeutic ways of going back, but chronic obstructive pulmonary disease (COPD) and asthma have a permanently active state. MiRNAs are changed in resolved inflammation, therefore let-7b is increased, modifying Toll response changes. Patient becomes less responsive to bacteria – patients are retuned to not die when challenged, but have also lost miRNAs. The basement membrane is normally very thin but becomes permanently changed (Burgstaller et al., 2017, Eur Respir J. 2017 doi: 10.1183/13993003. PMID: 28679607). The tissue is changed, so not just immune effects.

Hyaluranon is a major constituent of the inflamed lung. Is the lung inflamed or is it just because there are a lot of retained immune cells? The latter. Why does matrix persist? Hyaluronic acid synthase is increased (clearance unchanged, hyaluronase unchanged). Matrix turnover means that there are more activated immune cells. The impact of viral infections on lung matrix affects its mechanical stability and structural support. The composition of matrix also indirectly controls inflammation by influencing cell adhesion, migration, survival, proliferation and differentiation. Hyaluronan is a significant component of the lung extracellular matrix and production and degradation must be carefully balanced. Tracy discovered an imbalance in hyaluronan production following resolution of a severe lung influenza virus infection, driven by hyaluronan synthase 2 from epithelial cells, endothelial cells and fibroblasts. Furthermore hyaluronan, due to elevated TNF, sequesters CD44-expressing macrophages. Intranasal hyaluronidase reduces lung hyaluronan restoring function. Hyaluronidase is already used clinically. Digestion of HA restores lung function. Hyaluronidase (available for clinical trials) appears an interesting option. (Hussell T et al., Eur Respir Rev., 2018, doi: 10.1183/16000617.0032-2018. PMID: 29950305).

In cancer the matrix is abnormal, and the immune system is paralysed. In many instances the matrix is stiff. It may be possible to think differently about reactivating the immune system here.

In severe cell death, macrophages clear apoptotic cells, TAMs recognise external phosphatidyl serine. Reverse signalling to STAT1 turning off inflammation, so while apoptosis is going on the tissue is vulnerable to bacterial attack. Axl receptor only in lung so this represents a unique opportunity for targeted therapy. Changed matrix, lost apoptosis, but poorly cleared even if immunity suppressed.

Eight days after influenza, basal cells proliferate requiring Axl receptor to show that damage has happened. Basal cell show hyperplasia if apoptosis continues – most lung disease has this as a hallmark. Axl antagonists allows faster repair. Type 1 interferons (IFNs): Neuropeptide receptors alter away homeostasis Gfra2 part of GDNF family massively increased on lung TAMs. Needs to co-stimulate with ret which is induced by type 1 IFNs. The virus may act via TLR7/8, in epithelium. MMP2 is specifically increased to degrade collagen type IV to degrade basement membrane. Two chemokines bind CXCR2. CCL18 macrophages that drive resolution, no homologue in rodents.

Is COPD then associated with inflamed tissues or trapped inflammatory cells? Possibly like tumours. Will check-point inhibitors also release antigen presenting cells? New therapeutic options await validation.

Slide Downloads: PPTX | PDF


tonyharmar

Anthony Harmar, Professor of Pharmacology in Edinburgh.

In addition to being a brilliant pharmacologist, Tony set up the IUPHAR Edinburgh database group which initiated IUPHAR-DB, which expanded into the IUPHAR/BPS Guide to Pharmacology database (www.guidetopharmacology.org).

 

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