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.

GLP-1 increases heart rate by a direct action on the sinus node.

AIMS: Glucagon-like peptide-1 receptor agonists (GLP-1 RAs) are increasingly used to treat type 2 diabetes and obesity. Albeit cardiovascular outcomes generally improve, treatment with GLP-1 RAs is associated with increased heart rate, the mechanism of which is unclear. METHODS AND RESULTS: We employed a large animal model, the female landrace pig, and used multiple in-vivo and ex-vivo approaches including pharmacological challenges, electrophysiology and high-resolution mass spectrometry to explore how GLP-1 elicits an increase in heart rate. In anaesthetized pigs, neither cervical vagotomy, adrenergic blockers (alpha, beta or combined alpha-beta blockade), ganglionic blockade (hexamethonium) nor inhibition of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels (ivabradine) abolished the marked chronotropic effect of GLP-1. GLP-1 administration to isolated perfused pig hearts also increased heart rate, which was abolished by GLP-1 receptor blockade. Electrophysiological characterization of GLP-1 effects in vivo and in isolated perfused hearts localized electrical modulation to the atria and conduction system. In isolated sinus nodes, GLP-1 administration shortened action potential cycle length of pacemaker cells and shifted the site of earliest activation. The effect was independent of HCN blockade. Collectively, these data support a direct effect of GLP-1 on GLP-1 receptors within the heart. Consistently, single nucleus RNA sequencing (snRNAseq) showed GLP-1 receptor expression in porcine pacemaker cells. Quantitative phosphoproteomics analyses of sinus node samples revealed that GLP-1 administration leads to phosphorylation changes of calcium cycling proteins of the sarcoplasmic reticulum, known to regulate heart rate. CONCLUSION: GLP-1 has direct chronotropic effects on the heart mediated by GLP-1 receptors in pacemaker cells of the sinus node, inducing changes in action potential morphology and the leading pacemaker site through a calcium signaling response characterized by PKA-dependent phosphorylation of Ca2+ cycling proteins involved in pace making. Targeting the pacemaker calcium clock may be a strategy to lower heart rate in GLP-1 RA recipients.

L-valine is a powerful stimulator of GLP-1 secretion in rodents and stimulates secretion through ATP-sensitive potassium channels and voltage-gated calcium channels.

BACKGROUND: We previously reported that, among all the naturally occurring amino acids, L-valine is the most powerful luminal stimulator of glucagon-like peptide 1 (GLP-1) release from the upper part of the rat small intestine. This makes L-valine an interesting target for nutritional-based modulation of GLP-1 secretion. However, the molecular mechanism of L-valine-induced secretion remains unknown. METHODS: We aimed to investigate the effect of orally given L-valine in mice and to identify the molecular details of L-valine stimulated GLP-1 release using the isolated perfused rat small intestine and GLUTag cells. In addition, the effect of L-valine on hormone secretion from the distal intestine was investigated using a perfused rat colon. RESULTS: Orally given L-valine (1 g/kg) increased plasma levels of active GLP-1 comparably to orally given glucose (2 g/kg) in male mice, supporting that L-valine is a powerful stimulator of GLP-1 release in vivo (P > 0.05). Luminal L-valine (50 mM) strongly stimulated GLP-1 release from the perfused rat small intestine (P < 0.0001), and inhibition of voltage-gated Ca2+-channels with nifedipine (10 µM) inhibited the GLP-1 response (P < 0.01). Depletion of luminal Na+ did not affect L-valine-induced GLP-1 secretion (P > 0.05), suggesting that co-transport of L-valine and Na+ is not important for the depolarization necessary to activate the voltage-gated Ca2+-channels. Administration of the KATP-channel opener diazoxide (250 µM) completely blocked the L-valine induced GLP-1 response (P < 0.05), suggesting that L-valine induced depolarization arises from metabolism and opening of KATP-channels. Similar to the perfused rat small intestine, L-valine tended to stimulate peptide tyrosine-tyrosine (PYY) and GLP-1 release from the perfused rat colon. CONCLUSIONS: L-valine is a powerful stimulator of GLP-1 release in rodents. We propose that intracellular metabolism of L-valine leading to closure of KATP-channels and opening of voltage-gated Ca2+-channels are involved in L-valine induced GLP-1 secretion.

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.

High incidence of imperforate vagina in ADGRA3-deficient mice.

BACKGROUND: Ten percent of the female population suffers from congenital abnormalities of the vagina, uterus, or oviducts, with severe consequences for reproductive and psychological health. Yet, the underlying causes of most of these malformations remain largely unknown. ADGRA3 (GPR125) is involved in WNT signaling and planar cell polarity, mechanisms vital to female reproductive tract development. Although ADGRA3 is a well-established spermatogonial stem cell marker, its role within the female urogenital system remains unclear. RESULTS: In this study, we found Adgra3 to be expressed throughout the murine female urogenital system, with higher expression pre-puberty than after sexual maturation. We generated a global Adgra3-/- mouse line and observed imperforate vagina in 44% of Adgra3-/- females, resulting in distension of the reproductive tract and infertility. Ovarian morphology, plasma estradiol, ovarian Cyp19a1, and vaginal estrogen receptor α (Esr1) expression were unaffected. However, compared to controls, a significantly lower bone mineral density was found in Adgra3-/- mice. Whereas vaginal opening in mice is an estrogen-dependent process, 17ß-estradiol treatment failed to induce vaginal canalization in Adgra3-/- mice. Furthermore, a marked reduction in vaginal and ovarian progesterone receptor expression was observed concomitant with an upregulation of apoptotic regulators Bcl2, Bid, and Bmf in adult Adgra3-/- females with a closed vagina. CONCLUSIONS: Our collective results shed new insights into the complex mechanisms by which the adhesion receptor ADGRA3 regulates distal vaginal tissue remodeling during vaginal canalization via altered sex hormone responsiveness and balance in apoptotic regulators. This highlights the potential of ADGRA3 as a target in diagnostic screening and/or therapy for obstructive vaginal malformations in humans.

Ligand entry pathways control the chemical space recognized by GPR183.

The G protein-coupled receptor GPR183 is a chemotactic receptor with an important function in the immune system and association with a variety of diseases. It recognizes ligands with diverse physicochemical properties as both the endogenous oxysterol ligand 7α,25-OHC and synthetic molecules can activate the G protein pathway of the receptor. To better understand the ligand promiscuity of GPR183, we utilized both molecular dynamics simulations and cell-based validation experiments. Our work reveals that the receptor possesses two ligand entry channels: one lateral between transmembrane helices 4 and 5 facing the membrane, and one facing the extracellular environment. Using enhanced sampling, we provide a detailed structural model of 7α,25-OHC entry through the lateral membrane channel. Importantly, the first ligand recognition point at the receptor surface has been captured in diverse experimentally solved structures of different GPCRs. The proposed ligand binding pathway is supported by in vitro data employing GPR183 mutants with a sterically blocked lateral entrance, which display diminished binding and signaling. In addition, computer simulations and experimental validation confirm the existence of a polar water channel which might serve as an alternative entrance gate for less lipophilic ligands from the extracellular milieu. Our study reveals knowledge to understand GPR183 functionality and ligand recognition with implications for the development of drugs for this receptor. Beyond, our work provides insights into a general mechanism GPCRs may use to respond to chemically diverse ligands.

Amantadine for COVID-19 treatment (ACT) study: a randomized, double-blinded, placebo-controlled clinical trial.

OBJECTIVES: The COVID-19 pandemic has revealed a severe need for effective antiviral treatment. The objectives of this study were to assess if pre-emptive treatment with amantadine for COVID-19 in non-hospitalized persons ≥40 years or adults with comorbidities was able to prevent disease progression and hospitalization. Primary outcomes were clinical status on day 14. METHODS: Between 9 June 2021 and 27 January 2022, this randomized, double-blinded, placebo-controlled, single-centre clinical trial included 242 subjects with a follow-up period of 90 days. Subjects were randomly assigned 1:1 to either amantadine 100 mg or placebo twice daily for 5 days. The inclusion criteria were confirmed SARS-CoV-2 infection and at least one of (a) age ≥40 years, age ≥18 years and (b) at least one comorbidity, or (c) body mass index ≥30. The study protocol was published at www. CLINICALTRIALS: gov (unique protocol #02032021) and at www.clinicaltrialregister.eu (EudraCT-number 2021-001177-22). RESULTS: With 121 participants in each arm, we found no difference in the primary endpoint with 82 participants in the amantadine arm, and 92 participants in the placebo arm with no limitations to activities, respectively, and 25 and 37 with limitations to activities in the amantadine arm and the placebo arm, respectively. No participants in either group were admitted to hospital or died. The OR of having state severity increased by 1 in the amantadine group versus placebo was 1.8 (CI 1.0-3.3, [p 0.051]). On day 7, one participant was hospitalized in each group; throughout the study, this increased to five and three participants for amantadine versus placebo treatment (p 0.72). Similarly, on day 7, there was no difference in the status of oropharyngeal swabs. Most participants (108 in each group) were SARS-CoV-2 RNA positive (p 0.84). CONCLUSION: We found no effect of amantadine on disease progression of SARS-CoV-2 infection.

Glycine: a long-sought novel ligand for GPR158.

G-protein-coupled receptors (GPCRs) are important drug targets with chemically diverse ligands and varying intracellular coupling partners. Recent work by Laboute et al. deorphanized GPR158 as a metabotropic glycine receptor (mGlyR), thereby providing evidence of a novel neuromodulatory system involving this non-canonical Class C receptor with an impact on cognition and affective states.

Biased GLP-2 agonist with strong G protein-coupling but impaired arrestin recruitment and receptor desensitization enhances intestinal growth in mice.

BACKGROUND AND PURPOSE: Glucagon-like peptide-2 (GLP-2) is secreted postprandially by enteroendocrine L-cells and stimulates growth of the gut and bone. One GLP-2 analogue is approved for short bowel syndrome (SBS). To improve therapeutic efficacy, we developed biased GLP-2 receptor (GLP-2R) agonists through N-terminal modifications. EXPERIMENTAL APPROACH: Variants with Ala and Trp substitutions of the first seven positions of GLP-2(1-33) were studied in vitro for affinity, G protein activation (cAMP accumulation), recruitment of ß-arrestin 1 and 2, and internalization of the human and mouse GLP-2R. The intestinotrophic actions of the most efficacious (cAMP) biased variant were examined in mice. KEY RESULTS: Ala substitutions had more profound effects than Trp substitutions. For both, alterations at positions 1, 3 and 6 most severely impaired activity. ß-arrestin recruitment was more affected than cAMP accumulation. Among Ala substitutions, [H1A], [D3A] and [F6A] impaired potency (EC50 ) for cAMP-accumulation >20-fold and efficacy (Emax ) to 48%-87%, and were unable to recruit arrestins. The Trp substitutions, [A2W], [D3W] and [G4W] were partial agonists (Emax of 46%-59%) with 1.7-12-fold decreased potencies in cAMP and diminished ß-arrestin recruitment. The biased variants, [F6A], [F6W] and [S7W] induced less GLP-2R internalization compared with GLP-2, which induced internalization in a partly arrestin-independent manner. In mice, [S7W] enhanced gut trophic actions with increased weight of the small intestine, increased villus height and crypt depth compared with GLP-2. CONCLUSION AND IMPLICATIONS: G protein-biased GLP-2R agonists with diminished receptor desensitization have superior intestinotrophic effects and may represent improved treatment of intestinal insufficiency including SBS.

GPR183 antagonism reduces macrophage infiltration in influenza and SARS-CoV-2 infection.

RATIONALE: Severe viral respiratory infections are often characterised by extensive myeloid cell infiltration and activation and persistent lung tissue injury. However, the immunological mechanisms driving excessive inflammation in the lung remain poorly understood. OBJECTIVES: To identify the mechanisms that drive immune cell recruitment in the lung during viral respiratory infections and identify novel drug targets to reduce inflammation and disease severity. METHODS: Preclinical murine models of influenza A virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. RESULTS: Oxidised cholesterols and the oxysterol-sensing receptor GPR183 were identified as drivers of monocyte/macrophage infiltration to the lung during influenza A virus (IAV) and SARS-CoV-2 infection. Both IAV and SARS-CoV-2 infection upregulated the enzymes cholesterol 25-hydroxylase (CH25H) and cytochrome P450 family 7 subfamily member B1 (CYP7B1) in the lung, resulting in local production of the oxidised cholesterols 25-hydroxycholesterol (25-OHC) and 7α,25-dihydroxycholesterol (7α,25-OHC). Loss-of-function mutation of Gpr183 or treatment with a GPR183 antagonist reduced macrophage infiltration and inflammatory cytokine production in the lungs of IAV- or SARS-CoV-2-infected mice. The GPR183 antagonist significantly attenuated the severity of SARS-CoV-2 infection and viral loads. Analysis of single-cell RNA-sequencing data on bronchoalveolar lavage samples from healthy controls and COVID-19 patients with moderate and severe disease revealed that CH25H, CYP7B1 and GPR183 are significantly upregulated in macrophages during COVID-19. CONCLUSION: This study demonstrates that oxysterols drive inflammation in the lung via GPR183 and provides the first preclinical evidence for the therapeutic benefit of targeting GPR183 during severe viral respiratory infections.

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.

Glucose-dependent insulinotropic polypeptide receptor antagonist treatment causes a reduction in weight gain in ovariectomised high fat diet-fed mice.

BACKGROUND AND PURPOSE: The incretin hormone, gastric inhibitory peptide/glucose-dependent insulinotropic polypeptide (GIP), secreted by the enteroendocrine K-cells in the proximal intestine, may regulate lipid metabolism and adiposity, but its exact role in these processes is unclear. EXPERIMENTAL APPROACH: We characterized in vitro and in vivo antagonistic properties of a novel GIP analogue, mGIPAnt-1. We further assessed the in vivo pharmacokinetic profile of this antagonist, as well as its ability to affect high-fat diet (HFD)-induced body weight gain in ovariectomised mice during an 8-week treatment period. KEY RESULTS: mGIPAnt-1 showed competitive antagonistic properties to the GIP receptor in vitro as it inhibited GIP-induced cAMP accumulation in COS-7 cells. Furthermore, mGIPAnt-1 was capable of inhibiting GIP-induced glucoregulatory and insulinotropic effects in vivo and has a favourable pharmacokinetic profile with a half-life of 7.2 h in C57Bl6 female mice. Finally, sub-chronic treatment with mGIPAnt-1 in ovariectomised HFD mice resulted in a reduction of body weight and fat mass. CONCLUSION AND IMPLICATIONS: mGIPAnt-1 successfully inhibited acute GIP-induced effects in vitro and in vivo and sub-chronically induces resistance to HFD-induced weight gain in ovariectomised mice. Our results support the development of GIP antagonists for the therapy of obesity.

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.

A Blunted GPR183/Oxysterol Axis During Dysglycemia Results in Delayed Recruitment of Macrophages to the Lung During Mycobacterium tuberculosis Infection.

BACKGROUND: We previously reported that reduced GPR183 expression in blood from tuberculosis (TB) patients with diabetes is associated with more severe TB. METHODS: To further elucidate the role of GPR183 and its oxysterol ligands in the lung, we studied dysglycemic mice infected with Mycobacterium tuberculosis (Mtb). RESULTS: We found upregulation of the oxysterol-producing enzymes CH25H and CYP7B1 and increased concentrations of 25-hydroxycholesterol upon Mtb infection in the lungs of mice. This was associated with increased expression of GPR183 indicative of oxysterol-mediated recruitment of GPR183-expressing immune cells to the lung. CYP7B1 was predominantly expressed by macrophages in TB granulomas. CYP7B1 expression was significantly blunted in lungs from dysglycemic animals, which coincided with delayed macrophage infiltration. GPR183-deficient mice similarly had reduced macrophage recruitment during early infection. CONCLUSIONS: Taken together, we demonstrate a requirement of the GPR183/oxysterol axis for positioning of macrophages to the site of infection and add an explanation to more severe TB in diabetes patients.

Selective Boosting of CCR7-Acting Chemokines; Short Peptides Boost Chemokines with Short Basic Tails, Longer Peptides Boost Chemokines with Long Basic Tails.

The chemokine receptor CCR7 and its ligands CCL19 and CCL21 regulate the lymph node homing of dendritic cells and naïve T-cells and the following induction of a motile DC-T cell priming state. Although CCL19 and CCL21 bind CCR7 with similar affinities, CCL21 is a weak agonist compared to CCL19. Using a chimeric chemokine, CCL19CCL21N-term|C-term, harboring the N-terminus and the C-terminus of CCL21 attached to the core domain of CCL19, we show that these parts of CCL21 act in a synergistic manner to lower ligand potency and determine the way CCL21 engages with CCR7. We have published that a naturally occurring basic C-terminal fragment of CCL21 (C21TP) boosts the signaling of both CCL19 and CCL21. Boosting occurs as a direct consequence of C21TP binding to the CCR7 N-terminus, which seems to free chemokines with basic C-termini from an unfavorable interaction with negatively charged posttranslational modifications in CCR7. Here, we confirm this using a CCL19-variant lacking the basic C-terminus. This variant displays a 22-fold higher potency at CCR7 compared to WT CCL19 and is highly unaffected by the presence of C21TP. WT CCL19 has a short basic C-terminus, CCL21 a longer one. Here, we propose a way to differentially boost CCL19 and CCL21 activity as short and long versions of C21TP boost CCL19 activity, whereas only a long C21TP version can boost chemokines with a full-length CCL21 C-terminus.

GPR183 Is Dispensable for B1 Cell Accumulation and Function, but Affects B2 Cell Abundance, in the Omentum and Peritoneal Cavity.

B1 cells constitute a specialized subset of B cells, best characterized in mice, which is abundant in body cavities, including the peritoneal cavity. Through natural and antigen-induced antibody production, B1 cells participate in the early defense against bacteria. The G protein-coupled receptor 183 (GPR183), also known as Epstein-Barr virus-induced gene 2 (EBI2), is an oxysterol-activated chemotactic receptor that regulates migration of B cells. We investigated the role of GPR183 in B1 cells in the peritoneal cavity and omentum. B1 cells expressed GPR183 at the mRNA level and migrated towards the GPR183 ligand 7α,25-dihydroxycholesterol (7α,25-OHC). GPR183 knock-out (KO) mice had smaller omenta, but with normal numbers of B1 cells, whereas they had fewer B2 cells in the omentum and peritoneal cavity than wildtype (WT) mice. GPR183 was not responsible for B1 cell accumulation in the omentum in response to i.p. lipopolysaccharide (LPS)-injection, in spite of a massive increase in 7α,25-OHC levels. Lack of GPR183 also did not affect B1a- or B1b cell-specific antibody responses after vaccination. In conclusion, we found that GPR183 is non-essential for the accumulation and function of B1 cells in the omentum and peritoneal cavity, but that it influences the abundance of B2 cells in these compartments.

GIP and GLP-2 together improve bone turnover in humans supporting GIPR-GLP-2R co-agonists as future osteoporosis treatment.

The intestinal hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-2 (GLP-2) are key regulators of postprandial bone turnover in humans. We hypothesized that GIP and GLP-2 co-administration would provide stronger effect on bone turnover than administration of the hormones separately, and tested this using subcutaneous injections of GIP and GLP-2 alone or in combination in humans. Guided by these findings, we designed series of GIPR-GLP-2R co-agonists as template for new osteoporosis treatment. The clinical experiment was a randomized cross-over design including 10 healthy men administered subcutaneous injections of GIP and GLP-2 alone or in combination. The GIPR-GLP-2R co-agonists were characterized in terms of binding and activation profiles on human and rodent GIP and GLP-2 receptors, and their pharmacokinetic (PK) profiles were improved by dipeptidyl peptidase-4 protection and site-directed lipidation. Co-administration of GIP and GLP-2 in humans resulted in an additive reduction in bone resorption superior to each hormone individually. The GIPR-GLP-2R co-agonists, designed by combining regions of importance for cognate receptor activation, obtained similar efficacies as the two native hormones and nanomolar potencies on both human receptors. The PK-improved co-agonists maintained receptor activity along with their prolonged half-lives. Finally, we found that the GIPR-GLP-2R co-agonists optimized toward the human receptors for bone remodeling are not feasible for use in rodent models. The successful development of potent and efficacious GIPR-GLP-2R co-agonists, combined with the improved effect on bone metabolism in humans by co-administration, support these co-agonists as a future osteoporosis treatment.

Amantadine has potential for the treatment of COVID-19 because it inhibits known and novel ion channels encoded by SARS-CoV-2.

The dire need for COVID-19 treatments has inspired strategies of repurposing approved drugs. Amantadine has been suggested as a candidate, and cellular as well as clinical studies have indicated beneficial effects of this drug. We demonstrate that amantadine and hexamethylene-amiloride (HMA), but not rimantadine, block the ion channel activity of Protein E from SARS-CoV-2, a conserved viroporin among coronaviruses. These findings agree with their binding to Protein E as evaluated by solution NMR and molecular dynamics simulations. Moreover, we identify two novel viroporins of SARS-CoV-2; ORF7b and ORF10, by showing ion channel activity in a X. laevis oocyte expression system. Notably, amantadine also blocks the ion channel activity of ORF10, thereby providing two ion channel targets in SARS-CoV-2 for amantadine treatment in COVID-19 patients. A screen of known viroporin inhibitors on Protein E, ORF7b, ORF10 and Protein 3a from SARS-CoV-2 revealed inhibition of Protein E and ORF7b by emodin and xanthene, the latter also blocking Protein 3a. This illustrates a general potential of well-known ion channel blockers against SARS-CoV-2 and specifically a dual molecular basis for the promising effects of amantadine in COVID-19 treatment. We therefore propose amantadine as a novel, cheap, readily available and effective way to treat COVID-19.