Ligand-Specific Factors Influencing GLP-1 Receptor Post-Endocytic Trafficking and Degradation in Pancreatic Beta Cells.

The glucagon-like peptide-1 receptor (GLP-1R) is an important regulator of blood glucose homeostasis. Ligand-specific differences in membrane trafficking of the GLP-1R influence its signalling properties and therapeutic potential in type 2 diabetes. Here, we have evaluated how different factors combine to control the post-endocytic trafficking of GLP-1R to recycling versus degradative pathways. Experiments were performed in primary islet cells, INS-1 832/3 clonal beta cells and HEK293 cells, using biorthogonal labelling of GLP-1R to determine its localisation and degradation after treatment with GLP-1, exendin-4 and several further GLP-1R agonist peptides. We also characterised the effect of a rare GLP1R coding variant, T149M, and the role of endosomal peptidase endothelin-converting enzyme-1 (ECE-1), in GLP1R trafficking. Our data reveal how treatment with GLP-1 versus exendin-4 is associated with preferential GLP-1R targeting towards a recycling pathway. GLP-1, but not exendin-4, is a substrate for ECE-1, and the resultant propensity to intra-endosomal degradation, in conjunction with differences in binding affinity, contributes to alterations in GLP-1R trafficking behaviours and degradation. The T149M GLP-1R variant shows reduced signalling and internalisation responses, which is likely to be due to disruption of the cytoplasmic region that couples to intracellular effectors. These observations provide insights into how ligand- and genotype-specific factors can influence GLP-1R trafficking.

Protocol for kinetic mode potassium channel assays on common plate readers and microscopes.

Fluorescence-based potassium channel assays are typically run on expensive, hard to obtain, fluorescence imaging kinetic plate readers that are uncommon in most laboratories. Here we describe the use of the Brilliant Thallium Snapshot assay to conduct an endpoint potassium channel assay, so that it can be used across multiple plate reader platforms that are more common in many labs. These methods will allow users to identify modulators of potassium channels. For this work, we have taken a kinetic mode Molecular Devices FLIPR based protocol and adapted it to be utilized on endpoint plate readers, such as the BMG Labtech PHERAstar, to identify activators of GIRK channels in CHO cells. We demonstrate that both plate readers are functionally competent at generating excellent Z' values which makes them ideally suited to finding corollary hits from the Sigma LOPAC 1,280 screening collection. Importantly, this assay has also been validated using a high content reader, demonstrating the possibility of spatially resolving signals from individual cells within a mixed cell population. The compendium of these results shows the flexibility, accessibility and functionality of endpoint-compatible potassium channel assay readouts on more common plate readers.

Discovery and Characterization of Novel CNS-Penetrant GPR55 Agonists.

From our NETSseq-derived human brain transcriptomics data, we identified GPR55 as a potential molecular target for the treatment of motor symptoms in patients with Parkinson's disease. From a high-throughput screen, we identified and optimized agonists with nanomolar potency against both human and rat GPR55. We discovered compounds with either strong or limited ß-arrestin signaling and receptor desensitization, indicating biased signaling. A compound that showed minimal GPR55 desensitization demonstrated a reduction in firing frequency of medium spiny neurons cultured from rat striatum but did not reverse motor deficits in a rat hypolocomotion model. Further profiling of several desensitizing and non-desensitizing lead compounds showed that they are selective over related cannabinoid receptors CB1 and CB2 and that unbound brain concentrations well above the respective GPR55 EC50 can be readily achieved following oral administration. The novel brain-penetrant GPR55 agonists disclosed can be used to probe the role of this receptor in the brain.

A glucagon analogue decreases body weight in mice via signalling in the liver.

Glucagon receptor agonists show promise as components of next generation metabolic syndrome pharmacotherapies. However, the biology of glucagon action is complex, controversial, and likely context dependent. As such, a better understanding of chronic glucagon receptor (GCGR) agonism is essential to identify and mitigate potential clinical side-effects. Herein we present a novel, long-acting glucagon analogue (GCG104) with high receptor-specificity and potent in vivo action. It has allowed us to make two important observations about the biology of sustained GCGR agonism. First, it causes weight loss in mice by direct receptor signalling at the level of the liver. Second, subtle changes in GCG104-sensitivity, possibly due to interindividual variation, may be sufficient to alter its effects on metabolic parameters. Together, these findings confirm the liver as a principal target for glucagon-mediated weight loss and provide new insights into the biology of glucagon analogues.

Randomized, double-blind study of the safety, tolerability, and efficacy of tafenoquine versus mefloquine for malaria prophylaxis in nonimmune subjects.

This study represents the first phase III trial of the safety, tolerability, and effectiveness of tafenoquine for malaria prophylaxis. In a randomized (3:1), double-blinded study, Australian soldiers received weekly malaria prophylaxis with 200 mg tafenoquine (492 subjects) or 250 mg mefloquine (162 subjects) for 6 months on a peacekeeping deployment to East Timor. After returning to Australia, tafenoquine-receiving subjects received a placebo and mefloquine-receiving subjects received 30 mg primaquine daily for 14 days. There were no clinically significant differences between hematological and biochemical parameters of the treatment groups. Treatment-related adverse events for the two groups were similar (tafenoquine, 13.4%; mefloquine, 11.7%). Three subjects on tafenoquine (0.6%) and none on mefloquine discontinued prophylaxis because of possible drug-related adverse events. No diagnoses of malaria occurred for either group during deployment, but 4 cases (0.9%) and 1 case (0.7%) of Plasmodium vivax infection occurred among the tafenoquine and mefloquine groups, respectively, up to 20 weeks after discontinuation of medication. In a subset of subjects recruited for detailed safety assessments, treatment-related mild vortex keratopathy was detected in 93% (69 of 74) of tafenoquine subjects but none of the 21 mefloquine subjects. The vortex keratopathy was not associated with any effect on visual acuity and was fully resolved in all subjects by 1 year. Tafenoquine appears to be safe and well tolerated as malaria prophylaxis. Although the volunteers' precise exposure to malaria could not be proven in this study, tafenoquine appears to be a highly efficacious drug for malaria prophylaxis.

Signalling, trafficking and glucoregulatory properties of glucagon-like peptide-1 receptor agonists exendin-4 and lixisenatide.

BACKGROUND AND PURPOSE: Amino acid substitutions at the N-termini of glucagon-like peptide-1 (GLP-1) receptor agonist peptides result in distinct patterns of intracellular signalling, sub-cellular trafficking and efficacy in vivo. Here, we to determine whether sequence differences at the ligand C-termini of clinically approved GLP-1 receptor agonists exendin-4 and lixisenatide lead to similar phenomena. EXPERIMENTAL APPROACH: Exendin-4, lixisenatide and N-terminally substituted analogues with biased signalling characteristics were compared across a range of in vitro trafficking and signalling assays in different cell types. Fluorescent ligands and new time-resolved FRET approaches were developed to study agonist behaviours at the cellular and sub-cellular level. Anti-hyperglycaemic and anorectic effects of each parent ligand and their biased derivatives were assessed in mice. KEY RESULTS: Lixisenatide and exendin-4 showed equal binding affinity, but lixisenatide was fivefold less potent for cAMP signalling. Both peptides induced extensive GLP-1 receptor clustering in the plasma membrane and were rapidly endocytosed, but the GLP-1 receptor recycled more slowly to the cell surface after lixisenatide treatment. These combined deficits resulted in reduced maximal sustained insulin secretion and reduced anti-hyperglycaemic and anorectic effects in mice with lixisenatide. N-terminal substitution of His1 by Phe1 to both ligands had favourable effects on their pharmacology, resulting in improved insulin release and lowering of blood glucose. CONCLUSION AND IMPLICATIONS: Changes to the C-terminus of exendin-4 affect signalling potency and GLP-1 receptor trafficking via mechanisms unrelated to GLP-1 receptor occupancy. These differences were associated with changes in their ability to control blood glucose and therefore may be therapeutically relevant.

Disconnect between signalling potency and in vivo efficacy of pharmacokinetically optimised biased glucagon-like peptide-1 receptor agonists.

OBJECTIVE: The objective of this study was to determine how pharmacokinetically advantageous acylation impacts on glucagon-like peptide-1 receptor (GLP-1R) signal bias, trafficking, anti-hyperglycaemic efficacy, and appetite suppression. METHODS: In vitro signalling responses were measured using biochemical and biosensor assays. GLP-1R trafficking was determined by confocal microscopy and diffusion-enhanced resonance energy transfer. Pharmacokinetics, glucoregulatory effects, and appetite suppression were measured in acute, sub-chronic, and chronic settings in mice. RESULTS: A C-terminally acylated ligand, [F1,G40,K41.C16 diacid]exendin-4, was identified that showed undetectable ß-arrestin recruitment and GLP-1R internalisation. Depending on the cellular system used, this molecule was up to 1000-fold less potent than the comparator [D3,G40,K41.C16 diacid]exendin-4 for cyclic AMP signalling, yet was considerably more effective in vivo, particularly for glucose regulation. CONCLUSIONS: C-terminal acylation of biased GLP-1R agonists increases their degree of signal bias in favour of cAMP production and improves their therapeutic potential.

The Influence of Peptide Context on Signaling and Trafficking of Glucagon-like Peptide-1 Receptor Biased Agonists.

Signal bias and membrane trafficking have recently emerged as important considerations in the therapeutic targeting of the glucagon-like peptide-1 receptor (GLP-1R) in type 2 diabetes and obesity. In the present study, we have evaluated a peptide series with varying sequence homology between native GLP-1 and exendin-4, the archetypal ligands on which approved GLP-1R agonists are based. We find notable differences in agonist-mediated cyclic AMP signaling, recruitment of ß-arrestins, endocytosis, and recycling, dependent both on the introduction of a His → Phe switch at position 1 and the specific midpeptide helical regions and C-termini of the two agonists. These observations were linked to insulin secretion in a beta cell model and provide insights into how ligand factors influence GLP-1R function at the cellular level.