Using LanM Enzymes to Modify Glucagon-Like Peptides 1 and 2 in E.coli.

Selective modification of peptides is often exploited to improve pharmaceutically relevant properties of bioactive peptides like stability, circulation time, and potency. In Nature, natural products belonging to the class of ribosomally synthesized and post-translationally modified peptides (RiPPs) are known to install a number of highly attractive modifications with high selectivity. These modifications are installed by enzymes guided to the peptide by corresponding leader peptides that are removed as the last step of biosynthesis. Here, we exploit leader peptides and their matching enzymes to investigate the installation of D-Ala post-translationally in a critical position in the hormones, glucagon-like peptides (GLP) 1 and 2. We also offer insight into how precursor peptide design can modulate the modification pattern achieved.

Molecular and in vivo phenotyping of missense variants of the human glucagon receptor.

Naturally occurring missense variants of G protein-coupled receptors with loss of function have been linked to metabolic disease in case studies and in animal experiments. The glucagon receptor, one such G protein-coupled receptor, is involved in maintaining blood glucose and amino acid homeostasis; however, loss-of-function mutations of this receptor have not been systematically characterized. Here, we observed fewer glucagon receptor missense variants than expected, as well as lower allele diversity and fewer variants with trait associations as compared with other class B1 receptors. We performed molecular pharmacological phenotyping of 38 missense variants located in the receptor extracellular domain, at the glucagon interface, or with previously suggested clinical implications. These variants were characterized in terms of cAMP accumulation to assess glucagon-induced Gαs coupling, and of recruitment of ß-arrestin-1/2. Fifteen variants were impaired in at least one of these downstream functions, with six variants affected in both cAMP accumulation and ß-arrestin-1/2 recruitment. For the eight variants with decreased Gαs signaling (D63ECDN, P86ECDS, V96ECDE, G125ECDC, R2253.30H, R3085.40W, V3686.59M, and R3787.35C) binding experiments revealed preserved glucagon affinity, although with significantly reduced binding capacity. Finally, using the UK Biobank, we found that variants with wildtype-like Gαs signaling did not associate with metabolic phenotypes, whereas carriers of cAMP accumulation-impairing variants displayed a tendency toward increased risk of obesity and increased body mass and blood pressure. These observations are in line with the essential role of the glucagon system in metabolism and support that Gαs is the main signaling pathway effecting the physiological roles of the glucagon receptor.

GIP-derived GIP receptor antagonists - a review of their role in GIP receptor pharmacology.

Surprisingly, agonists, as well as antagonists of the glucose-dependent insulinotropic polypeptide receptor (GIPR), are currently being used or investigated as treatment options for type 2 diabetes and obesity - and both, when combined with glucagon-like peptide 1 receptor (GLP-1R) agonism, enhance GLP-1-induced glycemia and weight loss further. This paradox raises several questions regarding not only the mechanisms of actions of GIP but also the processes engaged during the activation of both the GIP and GLP-1 receptors. Here, we provide an overview of studies of the properties and actions of peptide-derived GIPR antagonists, focusing on GIP(3-30)NH2, a naturally occurring N- and C-terminal truncation of GIP(1-42). GIP(3-30)NH2 was the first GIPR antagonist administered to humans. GIP(3-30)NH2 and a few additional antagonists, like Pro3-GIP, have been used in both in vitro and in vivo studies to elucidate the molecular and cellular consequences of GIPR inhibition, desensitization, and internalization and, at a larger scale, the role of the GIP system in health and disease. We provide an overview of these studies combined with recent knowledge regarding the effects of naturally occurring variants of the GIPR system and species differences within the GIP system to enhance our understanding of the GIPR as a drug target.

Altered desensitization and internalization patterns of rodent versus human glucose-dependent insulinotropic polypeptide (GIP) receptors. An important drug discovery challenge.

BACKGROUND AND PURPOSE: The gut hormone glucose-dependent insulinotropic polypeptide (GIP) signals via the GIP receptor (GIPR), resulting in postprandial potentiation of glucose-stimulated insulin secretion. The translation of results from rodent studies to human studies has been challenged by the unexpected effects of GIPR-targeting compounds. We, therefore, investigated the variation between species, focusing on GIPR desensitization and the role of the receptor C-terminus. EXPERIMENTAL APPROACH: The GIPR from humans, mice, rats, pigs, dogs and cats was studied in vitro for cognate ligand affinity, G protein activation (cAMP accumulation), recruitment of beta-arrestin and internalization. Variants of the mouse, rat and human GIPRs with swapped C-terminal tails were studied in parallel. KEY RESULTS: The human GIPR is more prone to internalization than rodent GIPRs. Despite similar agonist affinities and potencies for Gαs activation, especially, the mouse GIPR shows reduced receptor desensitization, internalization and beta-arrestin recruitment. Using an enzyme-stabilized, long-acting GIP analogue, the species differences were even more pronounced. 'Tail-swapped' human, rat and mouse GIPRs were all fully functional in their Gαs coupling, and the mouse GIPR regained internalization and beta-arrestin 2 recruitment properties with the human tail. The human GIPR lost the ability to recruit beta-arrestin 2 when its own C-terminus was replaced by the rat or mouse tail. CONCLUSIONS AND IMPLICATIONS: Desensitization of the human GIPR is dependent on the C-terminal tail. The species-dependent functionality of the C-terminal tail and the different species-dependent internalization patterns, especially between human and mouse GIPRs, are important factors influencing the preclinical evaluation of GIPR-targeting therapeutic compounds.

Characterization of genetic variants of GIPR reveals a contribution of ß-arrestin to metabolic phenotypes.

Incretin-based therapies are highly successful in combatting obesity and type 2 diabetes1. Yet both activation and inhibition of the glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) in combination with glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) activation have resulted in similar clinical outcomes, as demonstrated by the GIPR-GLP-1R co-agonist tirzepatide2 and AMG-133 (ref. 3) combining GIPR antagonism with GLP-1R agonism. This underlines the importance of a better understanding of the GIP system. Here we show the necessity of ß-arrestin recruitment for GIPR function, by combining in vitro pharmacological characterization of 47 GIPR variants with burden testing of clinical phenotypes and in vivo studies. Burden testing of variants with distinct ligand-binding capacity, Gs activation (cyclic adenosine monophosphate production) and ß-arrestin 2 recruitment and internalization shows that unlike variants solely impaired in Gs signalling, variants impaired in both Gs and ß-arrestin 2 recruitment contribute to lower adiposity-related traits. Endosomal Gs-mediated signalling of the variants shows a ß-arrestin dependency and genetic ablation of ß-arrestin 2 impairs cyclic adenosine monophosphate production and decreases GIP efficacy on glucose control in male mice. This study highlights a crucial impact of ß-arrestins in regulating GIPR signalling and overall preservation of biological activity that may facilitate new developments in therapeutic targeting of the GIPR system.

The Location of Missense Variants in the Human GIP Gene Is Indicative for Natural Selection.

The intestinal hormone, glucose-dependent insulinotropic polypeptide (GIP), is involved in important physiological functions, including postprandial blood glucose homeostasis, bone remodeling, and lipid metabolism. While mutations leading to physiological changes can be identified in large-scale sequencing, no systematic investigation of GIP missense variants has been performed. Here, we identified 168 naturally occurring missense variants in the human GIP genes from three independent cohorts comprising ~720,000 individuals. We examined amino acid changing variants scattered across the pre-pro-GIP peptide using in silico effect predictions, which revealed that the sequence of the fully processed GIP hormone is more protected against mutations than the rest of the precursor protein. Thus, we observed a highly species-orthologous and population-specific conservation of the GIP peptide sequence, suggestive of evolutionary constraints to preserve the GIP peptide sequence. Elucidating the mutational landscape of GIP variants and how they affect the structural and functional architecture of GIP can aid future biological characterization and clinical translation.

N-terminal alterations turn the gut hormone GLP-2 into an antagonist with gradual loss of GLP-2 receptor selectivity towards more GLP-1 receptor interaction.

BACKGROUND AND PURPOSE: To fully elucidate the regulatory role of the GLP-2 system in the gut and the bones, potent and selective GLP-2 receptor (GLP-2R) antagonists are needed. Searching for antagonist activity, we performed systematic N-terminal truncations of human GLP-2(1-33). EXPERIMENTAL APPROACH: COS-7 cells were transfected with the human GLP-2R and assessed for cAMP accumulation or competition binding using 125 I-GLP-2(1-33)[M10Y]. To examine selectivity, COS-7 cells expressing human GLP-1 or GIP receptors were assessed for cAMP accumulation. KEY RESULTS: Affinity of the N-terminally truncated GLP-2 peptides for the GLP-2 receptor decreased with reduced N-terminal peptide length (Ki 6.5-871 nM), while increasing antagonism appeared with inhibitory potencies (IC50 ) values from 79 to 204 nM for truncation up to GLP-2(4-33) and then declined. In contrast, truncation-dependent increases in intrinsic activity were observed from an Emax of only 20% for GLP-(2-33) up to 46% for GLP-2(6-33) at 1 µM, followed by a decline. GLP-2(9-33) had the highest intrinsic efficacy (Emax 65%) and no antagonistic properties. Moreover, with truncations up to GLP-2(8-33), a gradual loss in selectivity for the GLP-2 receptor appeared with increasing GLP-1 receptor (GLP-1R) inhibition (up to 73% at 1 µM). Lipidation of the peptides improved antagonism (IC50 down to 7.9 nM) for both the GLP-2 and the GLP-1R. CONCLUSION AND IMPLICATIONS: The N-terminus of GLP-2 is crucial for GLP-2R activity and selectivity. Our observations form the basis for the development of tool compounds for further characterization of the GLP-2 system.

Mutational Landscape of the Proglucagon-Derived Peptides.

Strong efforts have been placed on understanding the physiological roles and therapeutic potential of the proglucagon peptide hormones including glucagon, GLP-1 and GLP-2. However, little is known about the extent and magnitude of variability in the amino acid composition of the proglucagon precursor and its mature peptides. Here, we identified 184 unique missense variants in the human proglucagon gene GCG obtained from exome and whole-genome sequencing of more than 450,000 individuals across diverse sub-populations. This provides an unprecedented source of population-wide genetic variation data on missense mutations and insights into the evolutionary constraint spectrum of proglucagon-derived peptides. We show that the stereotypical peptides glucagon, GLP-1 and GLP-2 display fewer evolutionary alterations and are more likely to be functionally affected by genetic variation compared to the rest of the gene products. Elucidating the spectrum of genetic variations and estimating the impact of how a peptide variant may influence human physiology and pathophysiology through changes in ligand binding and/or receptor signalling, are vital and serve as the first important step in understanding variability in glucose homeostasis, amino acid metabolism, intestinal epithelial growth, bone strength, appetite regulation, and other key physiological parameters controlled by these hormones.

Access to Mammography Facilities and Detection of Breast Cancer by Screening Mammography: A GIS Approach.

OBJECTIVES: The objective of the study was to examine the association between access to mammography facilities and utilization of screening mammography in an urban population. METHODS: Data on female breast cancer cases were obtained from an extensive mammography surveillance project. Distance to mammography facilities was measured by using GIS, which was followed by measuring geographical access to mammography facilities using Floating Catchment Area (FCA) method (considering all available facilities within an arbitrary radius from the woman's residence by using Arc GIS 9.0 software). RESULTS: Of 2,024 women, 91.4% were Caucasian; age ranged from 25 to 98 years; most (95%) were non-Hispanic in origin. Logistic regression found age, family history, hormone replacement therapy, physician recommendation, and breast cancer stage at diagnosis to be significant predictors of having had a previous mammogram. Women having higher access to mammography facilities were less likely to have had a previous mammogram compared to women who had low access, considering all the facilities within 10 miles (OR=0.41, CI=0.22-0.76), 30 miles (OR=0.52, CI=0.29-0.91) and 40 miles (OR=0.51, CI=0.28-0.92) radiuses. CONCLUSIONS: Physical distance to mammography facilities does not necessarily predict utilization of mammogram and greater access does not assure greater utilizations, due to constraints imposed by socio economic and cultural barriers. Future studies should focus on measuring access to mammography facilities capturing a broader dimension of access considering qualitative aspect of facilities, as well as other travel impedances.