The cognitive improvement in Alzheimer’s disease patients brought about by treatment with acetyl cholinesterase inhibitors has been largely attributed to enhanced M1-muscarinic receptor signalling. Recently, however, studies with M1-receptor knockout mice and with more selective M1-receptor modulators have suggested that this receptor may not directly mediate learning and memory.

A team led by researchers at the University of Leicester has now suggested an alternative mechanism involving the M3-muscarinic receptor which is widely expressed in many brain regions, including the hippocampus. M3-receptor knockout mice were found to show a deficit in fear conditioning learning and memory. A knock-in mouse strain expressing a phosphorylation-deficient receptor also showed a deficit in fear conditioning learning, indicating that the learning process involves receptor phosphorylation. Agonist treatment and fear conditioning training led to phosphorylation of the M3-receptor in the hippocampus, confirming the importance of receptor phoshorylation on learning and memory. The phosphorylation-deficient receptor was expressed normally at the cell surface and was able to signal via the Gq/11 calcium pathway, but was uncoupled from phosphorylation-dependent processes such as receptor internalization and arrestin recruitment. The study, which is published in PNAS, suggests that an M3-receptor modulator that enhances phosphorylation/arrestin-dependent (non-G protein) signalling may be beneficial in treating cognitive disorders. ‘Biased’ ligands – those able to direct signalling of GPCRs selectively through the phosphorylation/arrestin-dependent pathway – have recently been described for a number of other GPCRs.

The presence of multiple redundant and compensatory pathways controlling energy homeostasis has, so far, limited the effectiveness of anti-obesity treatments and suggests that combination therapy may be the best approach for treating the worldwide obesity epidemic. Writing in the journal Cell Metabolism, researchers at Merck have now demonstrated a role for the orphan bombesin receptor subtype 3 (BRS-3) in controlling energy balance.

Using a selective BRS-3 agonist (Bag-1) and antagonist (Bantag-1), the team have established a role for BRS-3 in the regulation of food intake, metabolic rate, and body weight. Intracerebroventricular infusion of the peptide Bantag-1 led to higher food intake and a progressive increase in adipose mass and body weight whereas oral administration of Bag-1 increased metabolic rate and reduced food intake, adipose weight, and body weight. Prolonged high levels of brain receptor occupancy by agonist increased weight loss, suggesting a lack of tachyphylaxis.

As well as suggesting a potential new target for the treatment of obesity, the discovery of selective BRS-3 agonists and antagonists will allow investigation of the mechanisms by which BRS-3 regulates energy metabolism as well as exploration of other aspects of BRS-3 biology.

Osteoporosis is the result of an imbalance between bone resorption and bone formation in the constant matrix remodelling process that occurs in healthy bone. Bone remodelling is regulated by parathyroid hormone (PTH) which stimulates bone formation by binding to PTH receptors (PTH1R) on osteoblasts but also indirectly stimulates the formation of new osteoclasts, leading to increased bone resorption. Continuous elevation of PTH in hyperparathyroid disease leads to loss of bone mass and osteoporosis but intermittent elevation with daily injections of a recombinant form of PTH(1-34) results in net bone gain.

A new study led by researchers at Duke University Medical Center describes similar net bone gain in mice treated with the biased agonist (d-Trp¹²,Tyr³⁴)-PTH(7–34) (PTH-βarr), which activates β-arrestin signalling but not classical G protein signalling. β-arrestins regulate G protein-coupled receptors both by inhibiting classical signalling and by initiating distinct β-arrestin-mediated signalling. The interplay of these distinct signalling pathways largely determines the cellular consequences of drugs acting at G protein-coupled receptors. In wild-type mice, both PTH-βarr and PTH(1–34), which activates both arrestin and classical pathways, induced anabolic bone formation. In β-arrestin2–null mice, treatment with PTH(1–34) led to a smaller increase in bone mineral density than in wild-type mice and treatment with PTH-βarr had no effect. The β-arrestin pathway leads primarily to trabecular bone formation and does not stimulate bone resorption. The study provides evidence that selective agonism of the β-arrestin pathway can elicit an in vivo response distinct from that elicited by a non-selective agonist, and suggests that such ligands may have therapeutic potential.

The study is published in the inaugural issue of Science Translational Medicine.

The incretin, glucagon-like peptide-1 (GLP-1), is an intestinal hormone that stimulates production and release of insulin from pancreatic beta cells. Consequently there has been considerable interest in mimicking the activity of GLP-1 for treatment of metabolic disorders such as Type-II diabetes and obesity. There are currently two approved classes of drug that modulate GLP-1 activity: analogues of GLP-1 and inhibitors of dipeptidyl peptidase IV (DPPIV). Analogues of GLP-1, such as the 39-residue synthetic peptide, exenatide, activate the GLP-1 receptor but are resistant to proteolytic cleavage by DPPIV. The gliptins, such as sitagliptin, inhibit DPPIV, extending the half-life of the natural hormone.

Researchers at Ecole Polytechnique Fédérale de Lausanne, in collaboration with the University of Perugia and Intercept Pharmaceuticals, have now published data showing that stimulation of TGR5, a G-protein coupled receptor, leads to release of GLP-1 in obese mice. The same group had previously demonstrated that activation of TGR5 in brown adipose tissue and muscle by endogenous bile acids boosted energy expenditure and reversed diet-induced obesity in mice.

In the current work the researchers used a combination of genetic gain- and loss-of-function studies together with the TGR5 agonist, INT-777, to show the link between TGR5 signalling and GLP-1 secretion. In vitro experiments with INT-777 in enteroendocrine L-cells confirmed the induction of GLP-1 secretion and that this was linked to increased intracellular ATP/ADP ratio and a subsequent rise in intracellular calcium mobilization.

The study, published in the September 2^nd edition of Cell Metabolism, opens up a potential third route to modulation of GLP-1 activity and treatment of metabolic disorders.

Compounds acting at G-protein coupled receptors (GPCRs) form the largest class of drug molecules but, although the biochemical steps involved in GPCR signalling are known in some detail, the location of these events in space and time in living cells is still poorly understood. Scientists at Novartis reported earlier this year that S₁P₁ receptors bound to the ligand FTY720 continue to signal after internalisation, albeit by a different pathway from that used when the receptors are at the cell surface. Writing in the journal PLOS Biology, scientists in Italy and Germany have now reported that the thyroid-stimulating hormone (TSH) receptor continues signalling via cAMP even after internalisation. Ligand signalling through GPCRs was traditionally thought to take place exclusively at the cell membrane and involve coupling of receptors to G-proteins, activation of G-proteins and subsequent signalling via second messengers such as cAMP. Receptor internalisation was believed to contribute to signal termination by reducing the number of receptors present at the cell surface – although endocytosis was later shown to be important for receptor resensitisation. Once inside the cell, GPCRs were believed to stop signalling to second messengers, but the new study shows that the TSH receptor continues to stimulate cAMP production after internalisation.

The team isolated native, 3-D thyroid follicles from transgenic mice with ubiquitous expression of an inert fluorescent sensor for cAMP, and showed that, although the TSH receptors were rapidly internalised after ligand binding, they continued to stimulate cAMP production from inside the cells. The cAMP signalling by the internalised receptors appeared, however, to be somewhat different from that occurring at the plasma membrane since it was sustained, rather than rapidly reversible, and led to a different pattern of downstream signals. Thyroid follicles were chosen for the experiment since they are a particularly good model in which to study the spatiotemporal dynamics of cAMP signalling – they maintain the supra-cellular organisation, size, and polarisation of thyroid cells in vivo, and are a rare example of cells that are under the strict control of a GPCR and cAMP for virtually all their functions.

The study shows that GPCR signalling in a physiological setting may be more complex than previously supposed, with the sub-cellular localisation of the GPCR playing an important role in the duration and spatial pattern of downstream signals. It will be very interesting to discover whether a majority of endogenous ligands – or drug molecules – continue to signal via internalised receptors and, if so, whether intracellular signalling is inherently different from that which occurs at the cell surface.

Many groups developing cannabinoid receptor agonists for the treatment of pain have sought to identify selective CB2 receptor agonists to avoid the psychotropic effects associated with CB1 receptor agonists and a recent report in the journal Science may provide another reason to aim for selectivity. Although there is compelling evidence that cannabis and cannabis-based medicines can help reduce chronic pain, the new research suggests that endocannabinoids acting via the CB1 receptor can amplify and prolong pain rather than alleviate it.

The spinal cord contains both excitatory and inhibitory neurons which together regulate the transmission of information, including pain signals, to the brain. In ex vivo experiments using mouse spinal cord tissue, researchers found that the mixed CB1/CB2 receptor agonist, WIN 55212-2, reduced the amplitude of inhibitory postsynaptic currents, an effect that was reversed by the CB1 receptor antagonist/inverse agonist, AM251. In anaesthetised rats, local spinal application of AM251 reversed the increase in action potential firing triggered by mechanical stimulation in an area surrounding a capsaicin injection site on the hind paw and, in mice, intrathecal administration of AM251 also reversed mechanical sensitisation. Studies in knockout animals indicated that CB1 receptors on inhibitory dorsal root neurons, rather than those on primary nociceptors, were responsible for capsaicin-induced secondary hyperalgesia. Mechanical sensitisation could also be evoked by intrathecal injection of the CB1/CB2 agonist, CP 55,940.

In human volunteers, 10-day treatment with the CB1 receptor antagonist/inverse agonist, rimonabant, had no effect on acute pain ratings induced by electrical stimulation of the forearm, but reduced the sizes of hyperalgesic and allodynic skin areas to around 50% of pre-treatment values. The study reveals an unexpected role for endocannabinoids acting on CB1 receptors on neurons in the dorsal horn in diminishing inhibitory control of pain signals.

Another recently published study concluded that activation of spinal CB1 receptors by administration of the CB1 agonist, arachidonyl-2-chloroethylamine reduced bone cancer-related pain and that presynaptic inhibition may contribute to this analgesic effect.

Localisation of CB1 receptors and precise pathways of pain signalling may explain the differing effects of CB1 agonists in pain models and human patients.

The discovery of the cannabis receptors CB₁ and CB₂ and the subsequent discovery of anandamide (N-arachidonoylethanolamine), the first endogenous agonist of both receptors, provided a rationale for the known analgesic properties of Cannabis sativa. Anandamide is rapidly hydrolyzed and inactivated by the serine hydrolase, fatty acid amide hydrolase (FAAH), leading to the hypothesis that inhibition of FAAH would be an effective analgesic strategy. Writing in the journal Chemistry & Biology, scientists at the Scripps Research Institute and Pfizer Inc. have now described the discovery of PF-3845, a highly efficacious and selective inhibitor of FAAH. Mechanistic and structural studies confirmed that PF-3845 acts as a covalent inhibitor and carbamylates the active site serine of FAAH. Initial experiments showed that PF-3845 is 10 to 20 times more potent than other FAAH inhibitors and has superior pharmacokinetic properties. In animal studies, PF-3845 raised brain anandamide levels for up to 24 hours and produced a significant cannabinoid receptor-dependent reduction in inflammatory pain. Even if PF-3845 is not itself progressed to the clinic, the team believe that it will be a valuable pharmacological tool for further in vivo characterization of the endocannabinoid system.

Although cannabis has a long history of use in rituals, as a medicine and as a psychoactive agent, it wasn’t until the late 1980s that the first receptor for the major psychoactive component, Δ9-tetrahydrocannabinol, was identified. This receptor, which came to be known as the CB₁ receptor, is expressed mainly in the brain and is responsible for the psychotropic effects of cannabis. Shortly afterwards, a second receptor, the CB₂ receptor, was discovered on cells of the immune system. Since then, lipids such as anandamide and 2-arachidonoylglycerol have been shown to be endogenous ligands for cannabis receptors.

More recently, a peptide antagonist of the CB₁ receptor was identified, and now US and Brazilian scientists have discovered peptide agonists in the brains of mice. The antagonist, hemopressin (PVNFKFLSH), is a 9-amino acid residue peptide derived from α-hemoglobin and the newly discovered agonists are N-terminally extended variants of hemopressin, incorporating two or three extra amino acids. The peptide agonists were found to activate a signal transduction pathway distinct from that activated by the endocannabinoid, 2-arachidonoylglycerol, or the classic CB₁ agonist, Hu-210.

The study, which is published in the FASEB Journal, suggests an additional mode of regulation of endogenous cannabinoid receptor activity, and the authors hope that this could lead to the discovery of new drugs for managing pain, stimulating appetite and preventing cannabis abuse.

Cachexia affects many different chronically ill patient populations, including those with cancer. It results in loss of body weight, particularly of lean body mass (LBM), and is estimated to be responsible for over 20% of all cancer-related deaths. Currently, available drugs are ineffective, and new therapies are urgently needed. The anorexigenic peptide, α-melanocyte stimulating hormone (α-MSH), is believed to be crucially involved in the normal and pathologic regulation of food intake and it was speculated that blockade of its central physiological target, the melanocortin-4 receptor (MC4R), might provide a promising anti-cachexia treatment strategy. The idea is supported by animal studies with agouti-related protein (AgRP), the endogenous inverse agonist at the MC4 receptor, which was found to affect two hallmark features of cachexia: to increase food intake and to reduce energy expenditure.

In 1998, it was reported that MC4R mutations were associated with inherited human obesity. MC4R mutations have a prevalence of 1-2.5% in people with body mass indexes greater than 30, making it the most commonly known genetic defect predisposing people to obesity.

Researchers at Santhera Pharmaceuticals have now published results with orally available MC4R antagonists in an animal model of tumour-induced cachexia. Once daily oral administration of both compounds SNT207858 and SNT207707, starting the day after tumour implantation, significantly reduced the tumour induced weight loss. SNT207707 binds to the MC4 receptor with an affinity of 8 nM and shows a more than 200-fold selectivity vs. MC3 and MC5. SNT207858 is a 22 nM MC4 antagonist with a 170-fold selectivity vs. MC3 and a 40-fold selectivity versus MC5.

Full details of the study are published in the journal PLoS One.

Available treatments for Alzheimer’s disease offer relatively small symptomatic benefits and more effective treatments are much needed. β-Amyloid has been implicated in the pathogenesis of Alzheimer’s disease and much attention has focussed on inhibiting two enzymes responsible for production of this peptide, the β- and γ-secretases. Although potent inhibitors of both enzymes have been identified, achieving robust activity in animal models and progression to clinical studies has proved more challenging. Using a high-throughput functional genomics screen, scientists at VIB and Galapagos Pharmaceuticals have now identified a constitutively expressed orphan G protein-coupled receptor, GPR3, as another modulator of β-amyloid production. GPR3 is highly expressed in areas of the brain associated with Alzheimer’s disease and levels are elevated in brain tissue from people with sporadic Alzheimer’s disease. The researchers showed that blocking GPR3 prevented accumulation of β-amyloid, both in cell culture experiments and in a mouse model of Alzheimer’s disease.

Since G protein-coupled receptors have been relatively easy to exploit as drug targets, the scientists hope that GPR3 will prove to be a promising target for the treatment of Alzheimer’s disease.

The study is published in the February 13^th issue of the journal Science.