The insulin receptor is a membrane protein responsible for regulation of nutrient balance and therefore an attractive target in the treatment of diabetes and metabolic syndrome. Pharmacology of the insulin receptor involves two distinct mechanisms, (1) activation of the receptor by insulin mimetics that bind in the extracellular domain and (2) inhibition of the receptor tyrosine kinase enzymatic activity in the cytoplasmic domain. While a complete structural picture of the full-length receptor comprising the entire sequence covering extracellular, transmembrane, juxtamembrane and cytoplasmic domains is still elusive, recent progress through cryoelectron microscopy has made it possible to describe the initial insulin ligand binding events at atomistic detail. We utilize this opportunity to obtain structural insights into the pharmacology of the insulin receptor. To this end, we conducted a comprehensive docking study of known ligands to the new structures of the receptor. Through this approach, we provide an in-depth, structure-based review of human insulin receptor pharmacology in light of the new structures.

In the retina, mineralocorticoid receptor (MR), expressed in vessels, glial and neuronal cells, is mainly activated by glucocorticoids. Under pathological conditions, ocular MR expression and corticoids change, leading in most cases to MR overactivation. Experimental models using MR agonists or antagonists, administered systemically or intraocularly, acutely or chronically and transgenic models, allowed to identify the deleterious consequences of MR pathway overactivation. Among them, oxidative stress, inflammation, deregulation of hydro-ionic channels, alteration of choroidal vasculature, angiogenesis and cell death, are common to major retinal diseases. Specific MR antagonists showed efficacy in models of diabetic retinopathy, ischaemia, retinal and choroidal angiogenesis and in models of glaucoma. It is highly likely that MR antagonists will find a place in the therapeutic arsenal of age-related macular degeneration, diabetic retinopathy, glaucoma and in pachychoroid associated diseases. Their use in humans is still limited by the need of biomarkers of MR activation and specific ocular formulations.

Background: Glucagon-like peptide-2 (GLP-2) is a 33 amino acid pro-glucagon-derived hormone produced in the intestinal enteroendocrine L-cells with trophic actions on both the gut and bones. GLP-2(1-33) is cleaved by the ubiquitous protease dipeptidyl peptidase-4 (DPP-4), resulting in GLP-2(3-33) with competitive antagonistic properties on the GLP-2 receptor (GLP-2R). Here we present two new hGLP-2 radioligands with different pharmacodynamic profiles. Experimental Approach: The methionine in position 10 of GLP-2(1-33) was substituted with tyrosine to enable oxidative iodination with incorporation of the iodine isotope [125I]. Similar substitution was done in GLP-2(3-33), thereby creating two new radioligands; an agonist [125]-hGLP-2(1-33,M10Y) and an antagonist [125]-hGLP-2(3-33,M10Y). Both were characterized regarding competition binding, binding kinetics and target tissue autoradiography. Key results: High and similar binding affinities for the human GLP-2R were observed for [125I]-hGLP-2(1-33,M10Y) and [125I]-hGLP-2(3-33,M10Y) with KD values of 59.3 nM and 40.6 nM, respectively. The M10Y substitution did not change the functional properties of GLP-2(1-33) or GLP-2(3-33). The antagonist [125I]-hGLP-2(3-33,M10Y) had higher Bmax and faster on-rate for the hGLP-2R compared to the agonist [125I]-hGLP-2(1-33,M10Y). Using autoradiography in mice strong labeling was observed in subepithelial myofibroblasts (SEMF) and pancreas islet cells. Both radioligands were selective for the GLP-2R, except for a low affinity binding to the GLP-1R (IC50 of 130 and 330 nM, respectively) Conclusion and implications: We successfully developed two new high affinity radioligands for GLP-2R studies and identified SEMF and pancreatic islets as target for GLP-2. It is uncertain whether binding in the pancreas islets results from GLP-2R or GLP-1R binding.

Liver fibrosis induced by chronic hepatic injury remains as a major cause of morbidity and mortality worldwide. Identification of susceptibility/prognosis factors and new therapeutic tools for treating hepatic fibrotic disorders of various etiologies are urgent medical needs. Cortistatin is a neuropeptide with potent anti-inflammatory and anti-fibrotic activities in lung that binds to receptors that are expressed in liver fibroblasts and hepatic stellate cells. Here, we evaluated the capacity of cortistatin to regulate liver fibrosis. We initially found that hepatic expression of cortistatin inversely correlated with liver fibrosis grade in mice and humans with hepatic disorders. Cortistatin-deficient mice showed exacerbated signs of liver damage and fibrosis and increased mortality rates when challenged to hepatotoxic and cholestatic injury. Compared to wild-type mice, non-parenchymal liver cells isolated from cortistatin-deficient mice showed increased presence of cells with activated myofibroblast phenotypes and a differential genetic signature that is indicative of activated hepatic stellate cells and periportal fibroblasts and of myofibroblasts with active contractile apparatus. Cortistatin treatment reversed in vivo and in vitro these exaggerated fibrogenic phenotypes and protected from progression to severe liver fibrosis in response to hepatic injury. In conclusion, we identify cortistatin as an endogenous molecular break of liver fibrosis and its deficiency as a potential poor-prognosis marker for chronic hepatic disorders that course with fibrosis. Cortistatin-based therapies emerge as attractive strategies for ameliorating severe hepatic fibrosis.

Bruton’s tyrosine kinase (BTK) is a non-receptor kinase best known for its role in B lymphocyte development that is critical for proliferation, and survival of leukaemia cells in B cell malignancies. However, BTK is expressed in myeloid cells, particularly monocytes and macrophages where its inhibition has been reported to exhibit anti-inflammatory properties. Therefore, we explored the role of BTK on the migration of myeloid cells in vitro and in vivo. Using the zymosan induced peritonitis model of sterile inflammation we demonstrated that acute (1 h prior to zymosan) inhibition of BTK using a wide range of FDA (Ibrutinib and Acalabrutinib) and non-FDA approved inhibitors (ONO-4059, CNX-774, Olumatinib and LFM-A13) reduced neutrophil and monocyte recruitment. XID mice, which have a point mutation in the Btk gene had reduced neutrophil and monocyte recruitment to the peritoneum following zymosan challenge. To better understand the role of BTK in myeloid cell recruitment we investigated both chemotaxis and chemokine production in monocytes and macrophages. Pharmacological or genetic inhibition of BTK signalling substantially reduced human monocyte and murine macrophage chemotaxis to a range of chemoattractants (complement C5a and CCL2). We also demonstrated that inhibition of BTK in tissue resident macrophages significantly decreases chemokine secretion by reducing NF-kB activity and Akt signalling. Our work has identified a new role of BTK in regulating myeloid cell recruitment via two mechanisms, 1) reducing monocyte/macrophages’ ability to undergo chemotaxis, and 2) reducing chemokine secretion, via reduced NF-kB activity in tissue resident macrophages.

Fungal infections cause serious problems in many aspects of human life; especially infections by fungal species represent problems in immunocompromised patients. Current antifungal antibiotics target various metabolic pathways, predominantly the cell wall or cellular membrane. However, numerous compounds are available to combat fungal infections, their efficacy is far from being satisfactory and some of them display substantial toxicity. The emerging resistance represents a serious issue as well; thus, there is a considerable need for new anti-fungal compounds with lower toxicity and higher effectiveness. One of the unique antifungal antibiotics is sordarin, the only known compound that acts on the fungal translational machinery per se. It has been shown that sordarin inhibits protein synthesis at the elongation step of the translational cycle, acting on eukaryotic elongation-factor-2. In this review, we are aiming to deliver a robust scientific platform promoting the development of antifungal compounds, especially focusing on molecular action of sordarin.

Background and Purpose: Azithromycin (AZM) is a macrolide antibiotic with well-described anti-inflammatory properties. This study aimed to substantiate its treatment potential in rheumatoid arthritis (RA). Experimental Approach: Gene expression profiles were collected by RNA-sequencing and the effects of AZM were assessed in functional assays. In vitro and vivo assays for examining the blockade of glucose-regulated protein 78 (GRP78) actions by AZM: assays for defining the anti-inflammatory activity of AZM using fibroblast-like synoviocytes (FLSs) from RA patients as well as collagen-induced arthritis (CIA) in DBA/1 mice. Identification and characterization of the binding of AZM to GRP78 using drug affability responsive target stability assay, proteomics and cellular thermal shift assay. Detect AZM inhibition of GRP78 and dependence of AZM’s anti-arthritis activity on GRP78. Key Results: AZM reduced pro-inflammatory factor production, cell migration, invasion and chemo-attractive potential, enhanced apoptosis, thereby reducing the deleterious inflammatory response of RA FLSs in vitro. AZM ameliorated the severity of CIA lesions. Transcriptional analyses implied that AZM treatment causes impairments in signaling cascades associated with cholesterol and lipid biosynthetic process. GRP78 was isolated as a novel target of AZM. AZM-mediated activation of unfolded protein response (UPR) via inhibiting GRP78 activity is required not only for inducing the expression of C/EBP-homologous protein (CHOP), but also for activation of sterol-regulatory element binding protein (SREBP) and its targeted genes involved in cholesterol and lipid biosynthetic process. Further, deletion of GRP78 abolished AZM’s anti-arthritis activity. Conclusion and Implications: These findings confirmed that AZM is an anti-arthritis therapeutic drug for RA treatment.

Aldosterone binds to the mineralocorticoid receptor (MR), a transcription factor of the nuclear receptor family, present in the kidney and in various other non-epithelial cells including the heart and the vasculature (Cannavo et al., 2018). Indeed, extra-renal pathophysiological effects of this hormone have been characterized, extending its actions to the cardiovascular (CV) system (Messaoudi et al., 2012). A growing body of clinical and pre-clinical evidence suggests that MR activation plays an important pathophysiological role in CV remodeling by promoting cardiac hypertrophy, fibrosis, arterial stiffness, as well as in inflammation and oxidative stress (Bauersachs et al., 2015). The following review article outlines the role of MR in CV disease with a focus on myocardial remodeling and heart failure (HF) including clinical trials as well as cellular and animal studies.

Background and Purpose: Superoxide anions can reduce the bioavailability and actions of endothelium-derived NO. In human resistance-sized arteries, endothelium-dependent vasodilatation can be mediated by H2O2 instead of NO. We tested the hypotheses that in resistance arteries from patients with resistant cardiovascular disease (CVD), endothelium-dependent vasodilatation uses mechanisms that are either insensitive to oxidative stress or involve a reactive oxygen species. Experimental Approach: Small arteries were isolated from biopsies of the parietal pericardium of patients undergoing elective cardiothoracic surgery and were studied by immunohistochemical and organ chamber techniques. Key Results: NO-synthases 1, 2 and 3, superoxide dismutase 1 and catalase proteins were observed in the microvascular wall. Relaxing responses to bradykinin were endothelium dependent. During submaximal depolarization-induced contraction, these relaxations were inhibited by inhibitors of NO-synthases (NOS) and soluble guanylyl cyclase (sGC) but not by scavengers of NO or HNO, inhibitors of cyclooxygenases, neuronal NO-synthase, superoxide dismutase or catalase, or by exogenous catalase. During contraction stimulated by endothelin-1, these relaxations were not reduced by any of these interventions except DETCA which caused a small reduction. Conclusion and Implications: In resistance arteries from patients with resistant CVD, endothelium-dependent relaxations seem not to be mediated by NO, HNO or H2O2 although NOS and sGC can be involved. These vasodilator responses proceed during excessive oxidative stress.

Migraine is one of the most common of neurological disorders with a global prevalence of up to 15%. One in five migraineurs have frequent episodic or chronic migraine requiring prophylactic treatment. In recent years, specific pharmaceutical treatments targeting calcitonin gene-related peptide (CGRP) signalling molecules have provided safe and effective treatments; monoclonal antibodies for prophylaxis and gepants for acute therapy. Albeit the beneficious impact of these new drugs, it is important to understand the molecular mechanisms involved to better understand migraine pathophysiology and improve the therapy. Here we describe current views on the role of the CGRP family of peptides CGRP, calcitonin (CT), adrenomedullin (AM), amylin (AMY) and their receptors in the trigeminovascular system (TGV). All these molecules are present within the TGV system but differ in expression and localization. It is likely that they have different roles, which can be utilized in providing additional drug targets.

Cancer cachexia is one of the most common causes of death among cancer patients, no effective anti-cachectic treatment is currently available. In experimental cachectic animal models, aberrant activation of STAT3 in skeletal muscle has been found to contribute to muscle wasting. However, its clinical association, the factors regulating STAT3 activation, and the molecular mechanisms remain incompletely understood. Here, we show that an enhanced interaction between activated STAT3 and HSP90, which were observed in the skeletal muscle of cancer cachexia patients, is a crucial event for the development of cachectic muscle wasting. Administration of HSP90 inhibitors 17DMAG and PU-H71 alleviated the muscle wasting in C26 tumor-bearing cachectic mice or the myotube atrophy of C2C12 cells induced by C26 conditional medium. A mechanistic study indicated that in cachectic skeletal muscle, prolonged STAT3 activation transactivated FOXO1 by binding directly to its promoter and triggered the muscle wasting in a FOXO1-dependent manner; Our results demonstrate that the HSP90/STAT3/FOXO1 axis plays a critical role in the cachectic muscle wasting, which might serve as potential therapeutic target for the treatment of cancer cachexia.

Pyroptosis, is a specialized form of inflammatory cell death which aids the defensive response against invading pathogens. Its tight regulation is lost during infection by the severe acute respiratory coronavirus 2 (SARS-CoV-2) and thus uncontrolled pyroptosis disrupts the immune system and the integrity of organs defining the critical conditions in patients with high viral load. Molecular pathways engaged downstream to the formation and stabilization of the inflammasome -required to execute the process- have been uncovered and drugs are available for their regulation. On the contrary, pharmacological inferring of the upstream events -which are critical to sense and interpret the initial damage by the pathogen- is far from being elucidated. This limits our capacity to identify early markers and targets to ameliorate SARS-CoV-2 linked pyroptosis. Here we aim to raise attention on mitochondria and pathways leading to its dysfunction with the goal to inform early steps of inflammasome and devise tools to interpret and counteract diseases by the SARS-CoV-2.

Diabetes is a chronic metabolic disorder associated with the accelerated development of macrovascular (atherosclerosis, coronary artery disease) and microvascular complications (nephropathy, retinopathy and neuropathy), which remain the principal cause of mortality and morbidity in this population. Current understanding of cellular and molecular pathways of diabetes-driven vascular complications as well as therapeutic interventions have arisen from studying disease pathogenesis in animal models. Diabetes-associated vascular complications are multi-faceted, involving the interaction between various cellular and molecular pathways. Thus, the choice of an appropriate animal model to study vascular pathogenesis is important in our quest to identify innovative and mechanism-based targeted therapies to reduce the burden of diabetic complications. Herein, we provide up-to-date information on available mouse models of both Type 1 and Type 2 diabetic vascular complications as well as experimental analysis and research outputs.

- Invited Commentary –

Since the start of the novel coronavirus SARS-Cov-2 pandemic, a disease that has become one of the world’s greatest global health challenges, the role of the immune system has been at the forefront of scientific studies. The pathophysiology of COVID-19 is complex, which is evident by those at higher risk for poor outcome. Multiple systems contribute to thrombosis and inflammation seen in COVID-19 patients, including neutrophil dysfunction, platelet activation, endothelial cell activation. Understanding how the immune system functions in different patient cohorts (particularly given recent emerging events with the Oxford/AstraZeneca vaccine) is vital to understanding the pathophysiology of this devastating disease and for subsequent development of novel therapeutic targets and expedite possible drug repurposing strategies that could benefit society on a global scale.

Sepsis causes multi-organ dysfunction and is a major cause of death in intensive care units, but there are no treatments that reverse the pathophysiological effects of sepsis. Vitamin C has antioxidant, anti-inflammatory, anticoagulant and immune modulatory actions, so is a potential treatment for sepsis. Recent clinical trials of high-doses of intravenous vitamin C (6-16 g/day) had variable effects. Since much higher doses are without side-effects in cancer and burns patients, we studied the effects of a mega-dose of intravenous sodium ascorbate (150 g/40 kg) in a clinically relevant ovine model of sepsis. This treatment dramatically improved the clinical state and over 3-7-h improved cardiovascular, pulmonary, hepatic and renal function and reduced body temperature. In a critically ill COVID-19 patient, intravenous sodium ascorbate (60 g) restored arterial pressure, improved renal function and increased arterial blood oxygen levels. Clinical trials are testing the effectiveness of mega-dose vitamin C in septic patients.

Background and Purpose Glutamate receptor mediated enhanced excitatory neurotransmission is typically associated with mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS). Kynurenic acid (KYNA) and quinolinic acid (QUIN) are two important tryptophan-kynurenine pathway (KP) metabolites that modulate glutamate receptor activity. This study was designed to test the hypothesis that alteration in metabolism of KP metabolites in the hippocampus of patients with MTLE-HS contributes to abnormal glutamatergic transmission. Experimental Approach TKP metabolites level were determined using HPLC and LC-MS/MS in the hippocampal samples of patients with MTLE-HS compared to autopsy and non-seizure control samples. mRNA and protein expression of TKP enzymes were determined by qPCR and western blot. Spontaneous glutamatergic activities were recorded from pyramidal neurons in presence of kynurenine (KYN) and KYNA using whole cell patch clamp. Key Results We observed significantly reduced KYNA and elevated QUIN levels in the hippocampal samples, while KYN level remains unaltered. Spontaneous glutamatergic activity in the hippocampal samples was higher compared to that in non-seizure controls. Treatment with kynurenine inhibited the glutamatergic activity in non-seizure control samples but not in case of the hippocampal samples. However, exogenously applied KYNA inhibited glutamatergic activity in both non-seizure control and hippocampal samples. We also observed reduced levels of enzyme kynurenine aminotransferase II and its co-factor pyridoxal phosphate in the hippocampal samples. Conclusion Our findings indicate that altered metabolism of TKP metabolites in hippocampus could contribute to hyperglutamatergic tone in patients with MTLE-HS.

Background and Purpose: Many pain-triggering nociceptor neurons express TRPV1 or TRPA1, cation-selective channels with large pores that enable permeation of QX-314, a cationic analogue of lidocaine. Co-application of QX-314 with TRPV1 or TRPA1 activators can silence nociceptors. We now describe BW-031, a novel more potent cationic sodium channel inhibitor, test whether its application alone can inhibit the pain associated with tissue inflammation, and whether this strategy can also inhibit cough. Experimental Approach: We characterized BW-031 inhibition of sodium channels and tested BW-031 in three models of inflammatory pain: rat paw inflammation produced by Complete Freund’s Adjuvant injection or surgical incision and a mouse paw UV burn model. We also tested the ability of BW-031 to inhibit cough induced by inhalation of dilute citric acid in guinea pigs. Key Results: BW-031 inhibited Nav1.7 and Nav1.1 channels with ~6-fold greater potency than QX-314 when introduced inside cells and entered capsaicin-activated TRPV1 expressing sensory neurons. BW-031 inhibited inflammatory pain in all three models, producing more effective and longer-lasting inhibition of pain than QX-314 in the mouse UV burn model. BW-031 was also effective in reducing cough counts by 78-90% when applied intratracheally under isoflurane anesthesia or by aerosol inhalation in awake guinea pigs with airway inflammation produced by ovalbumin sensitization. Conclusion and Implications: BW-031 a novel cationic sodium channel inhibitor can be applied locally as a single agent to inhibit inflammatory pain and also effectively inhibits cough in a guinea pig model of nociceptor-activated cough, suggesting a new clinical approach to treating cough.