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Ionotropic glutamate receptors mediate most excitatory neurotransmission in the central nervous system and function by opening a transmembrane ion channel upon binding of glutamate. Despite their crucial role in neurobiology, the architecture and atomic structure of an intact ionotropic glutamate receptor are unknown. Here we report the crystal structure of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-sensitive, homotetrameric, rat GluA2 receptor at 3.6 Å resolution in complex with a competitive antagonist. The receptor harbours an overall axis of two-fold symmetry with the extracellular domains organized as pairs of local dimers and with the ion channel domain exhibiting four-fold symmetry. A symmetry mismatch between the extracellular and ion channel domains is mediated by two pairs of conformationally distinct subunits, A/C and B/D. Therefore, the stereochemical manner in which the A/C subunits are coupled to the ion channel gate is different from the B/D subunits. Guided by the GluA2 structure and site-directed cysteine mutagenesis, we suggest that GluN1 and GluN2A NMDA ( N -methyl- d -aspartate) receptors have a similar architecture, with subunits arranged in a 1-2-1-2 pattern. We exploit the GluA2 structure to develop mechanisms of ion channel activation, desensitization and inhibition by non-competitive antagonists and pore blockers. Glutamate receptor: structure of the ion channel that keeps neurons in touch Most of the excitatory neurotransmissions in the central nervous system, the events that allow neurons to 'talk' to each other, are mediated by ionotropic glutamate receptors that act by opening a transmembrane ion channel on binding glutamate. Little was known about their overall structure, but now Eric Gouaux and colleagues report the crystal structure of the homotetrameric AMPA-subtype rat GluA2 receptor bound to a competitive antagonist. The structure reveals a novel symmetry arrangement requiring two of the four subunits to adopt a different shape from the other two. This means that glutamate binding, and ensuing channel opening, is not the same for each subunit. The structure, taken with data from crystallographic and site-directed mutagenesis experiments, suggests that other glutamate receptor subtypes, including kainate and NMDA, have similar overall architectures and molecular symmetries. Mechanisms of ion channel activation, desensitization and inhibition by non-competitive antagonists and pore blockers can be inferred from this structure. The majority of excitatory neurotransmission in the central nervous system is mediated by ionotropic glutamate receptors, which function by opening a transmembrane ion channel upon binding of glutamate. However, despite this crucial role in neurobiology, the architecture and atomic structure of an intact isotropic glutamate receptor are unknown. The X-ray crystal structure of the rat GluA2 receptor in complex with a competitive antagonist is now reported and analysed.

Pharmacological inhibition of VEGF-A has proven to be effective in inhibiting angiogenesis and vascular leak associated with cancers and various eye diseases. However, little information is currently available on the binding kinetics and relative biological activity of various VEGF inhibitors. Therefore, we have evaluated the binding kinetics of two anti-VEGF antibodies, ranibizumab and bevacizumab, and VEGF Trap (also known as aflibercept), a novel type of soluble decoy receptor, with substantially higher affinity than conventional soluble VEGF receptors. VEGF Trap bound to all isoforms of human VEGF-A tested with subpicomolar affinity. Ranibizumab and bevacizumab also bound human VEGF-A, but with markedly lower affinity. The association rate for VEGF Trap binding to VEGF-A was orders of magnitude faster than that measured for bevacizumab and ranibizumab. Similarly, in cell-based bioassays, VEGF Trap inhibited the activation of VEGFR1 and VEGFR2, as well as VEGF-A induced calcium mobilization and migration in human endothelial cells more potently than ranibizumab or bevacizumab. Only VEGF Trap bound human PlGF and VEGF-B, and inhibited VEGFR1 activation and HUVEC migration induced by PlGF. These data differentiate VEGF Trap from ranibizumab and bevacizumab in terms of its markedly higher affinity for VEGF-A, as well as its ability to bind VEGF-B and PlGF.

Complement is a key component of the innate immune system. Inappropriate complement activation underlies the pathophysiology of a variety of diseases. Complement component 5 (C5) is a validated therapeutic target for complement-mediated diseases, but the development of new therapeutics has been limited by a paucity of preclinical models to evaluate the pharmacokinetic (PK) and pharmacodynamic (PD) properties of candidate therapies. The present report describes a novel humanized C5 mouse and its utility in evaluating a panel of fully human anti-C5 antibodies. Surprisingly, humanized C5 mice revealed marked differences in clearance rates amongst a panel of anti-C5 antibodies. One antibody, pozelimab (REGN3918), bound C5 and C5 variants with high affinity and potently blocked complement-mediated hemolysis in vitro. In studies conducted in both humanized C5 mice and cynomolgus monkeys, pozelimab demonstrated prolonged PK and durable suppression of hemolytic activity ex vivo. In humanized C5 mice, a switch in dosing from in-house eculizumab to pozelimab was associated with normalization of serum C5 concentrations, sustained suppression of hemolytic activity ex vivo, and no overt toxicity. Our findings demonstrate the value of humanized C5 mice in identifying new therapeutic candidates and treatment options for complement-mediated diseases.

Heart failure is a leading cause of morbidity and mortality 1 , 2 . Elevated intracardiac pressures and myocyte stretch in heart failure trigger the release of counter-regulatory natriuretic peptides, which act through their receptor (NPR1) to affect vasodilation, diuresis and natriuresis, lowering venous pressures and relieving venous congestion 3 – 8 . Recombinant natriuretic peptide infusions were developed to treat heart failure but have been limited by a short duration of effect 9 , 10 . Here we report that in a human genetic analysis of over 700,000 individuals, lifelong exposure to coding variants of the NPR1 gene is associated with changes in blood pressure and risk of heart failure. We describe the development of REGN5381, an investigational monoclonal agonist antibody that targets the membrane-bound guanylate cyclase receptor NPR1. REGN5381, an allosteric agonist of NPR1, induces an active-like receptor conformation that results in haemodynamic effects preferentially on venous vasculature, including reductions in systolic blood pressure and venous pressure in animal models. In healthy human volunteers, REGN5381 produced the expected haemodynamic effects, reflecting reductions in venous pressures, without obvious changes in diuresis and natriuresis. These data support the development of REGN5381 for long-lasting and selective lowering of venous pressures that drive symptomatology in patients with heart failure. Durable agonism of NPR1 achieved with a novel investigational monoclonal antibody could mirror the positive hemodynamic changes in blood pressure and heart failure identified in humans with lifelong exposure to NPR1 coding variants.

Ionotropic glutamate receptors (iGluRs) mediate the majority of excitatory neurotransmission in the central nervous system and function by opening a transmembrane ion channel upon binding of glutamate. Despite their crucial role in neurobiology, the architecture and atomic structure of an intact iGluR is unknown. Here we report the crystal structure of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-sensitive, homotetrameric, rat GluA2 receptor at 3.6 Å resolution in complex with a competitive antagonist. The receptor harbors an overall axis of 2-fold symmetry with the extracellular domains organized as pairs of local dimers and with the ion channel domain exhibiting 4-fold symmetry. A symmetry mismatch between the extracellular and ion channel domains is mediated by two pairs of conformationally distinct subunits, A/C and B/D. Therefore, the stereochemical manner in which the A/C subunits are coupled to the ion channel gate is different from the B/D subunits. Guided by the GluA2 structure and site-directed cysteine mutagenesis we suggest that GluN1/GluN2A NMDA (N-methyl-d-aspartate) receptors have a similar architecture with subunits arranged in a 1-2-1-2 pattern. We exploit the GluA2 structure to develop mechanisms of ion channel activation, desensitization and inhibition by non competitive antagonists and pore blockers.

Diphtheria toxin (DT) undergoes a low pH-induced conformational change which allows it to penetrate the endosomal membrane and translocate its catalytic (C) domain to the cytosol, leading to cell death. The largely α-helical transmembrane (T) domain of the toxin is believed to mediate these processes and is, therefore, the focus of intense study. In this work, the topography of the membrane-inserted T domain, with a concentration on the moderately hydrophobic helices 5-7, was elucidated using Cys-scanning mutagenesis. After labeling with a Cys-specific fluorescent probe, the polarity of the residue's environment, their accessibility to externally added antibodies, and their penetration into lipid bilayers were monitored using a variety of fluorescence and fluorescence quenching techniques. A novel topography assay was also developed to distinguish residues located on the cis and trans side of the bilayer by measuring the reactivity of biotinylated T domain Cys residues with externally added or vesicle-entrapped BODIPY-streptavidin. It was found that C-terminal helices 5-9 insert deeply into the membrane under similar conditions while the hydrophilic helices 1-4 lie close to the external surface. However, unlike helices 8–9, which form a stable transmembrane helical hairpin, helices 5–7 insert in a non-transmembraneous fashion with two distinctly inserted segments corresponding to the hydrophobic portions of helix 5 and 6/7. Additionally, covalent attachment of the toxin's A chain (C domain) to the N-terminus of the T domain had little effect on the observed behavior of helices 5–9. Comparison with the proposed topography of the T domain in the open channel state (Senzel et. al (2000), J. Gen. Physiol. 115, 421–434) suggests that the topography observed in this study may represent an early stage in a translocation process in which helices 5–7 play a dynamic role.