60 research outputs found

    Therapeutic Cleavage of Anti-Aquaporin-4 Autoantibody in Neuromyelitis Optica by an IgG-Selective Proteinase

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    ABSTRACT Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system caused by binding of pathogenic IgG autoantibodies (NMO-IgG) to astrocyte water channel aquaporin-4 (AQP4). Astrocyte damage and downstream inflammation require NMO-IgG effector function to initiate complement-dependent cytotoxicity (CDC) and antibodydependent cell-mediated cytotoxicity (ADCC). Here, we evaluated the potential therapeutic utility of the bacterial enzyme IdeS (IgG-degrading enzyme of Streptococcus pyogenes), which selectively cleaves IgG antibodies to yield Fc and F(ab9) 2 fragments. In AQP4-expressing cell cultures, IdeS treatment of monoclonal NMO-IgGs and NMO patient sera abolished CDC and ADCC, even when IdeS was added after NMO-IgG was bound to AQP4. Binding of NMO-IgG to AQP4 was similar to that of the NMO-F(ab9) 2 generated by IdeS cleavage. NMO-F(ab9) 2 competitively displaced pathogenic NMO-IgG, preventing cytotoxicity, and the Fc fragments generated by IdeS cleavage reduced CDC and ADCC. IdeS efficiently cleaved NMO-IgG in mice in vivo, and greatly reduced NMO lesions in mice administered NMO-IgG and human complement. IgGselective cleavage by IdeS thus neutralizes NMO-IgG pathogenicity, and yields therapeutic F(ab9) 2 and Fc fragments. IdeS treatment, by therapeutic apheresis or direct administration, may be beneficial in NMO

    TITLE PAGE Small-Molecule Vasopressin-2 Receptor Antagonist Identified by a G- Protein Coupled Receptor 'Pathway' Screen MOL #34496 2 RUNNING TITLE PAGE Running title: Small-molecule V 2 R antagonist Corresponding author: Number of text pages: 31 Number o

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    Number of references: 40 Number of words in Abstract: 200 Number of words in Introduction: 496 Number of words in Discussion: 1,102 List of abbreviations: GPCR, G-protein coupled receptor; cAMP, cyclic adenosine monophospate; CFTR

    Small-Molecule Vasopressin-2 Receptor Antagonist Identified by a G-Protein Coupled Receptor "Pathway" Screen

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    ABSTRACT G-protein-coupled receptors (GPCRs) such as the vasopressin-2 receptor (V 2 R) are an important class of drug targets. We developed an efficient screen for GPCR-induced cAMP elevation using as read-out cAMP activation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl Ϫ channels. Fischer rat thyroid cells expressing CFTR and a halide-sensing yellow fluorescent protein (H148Q/I152L) were transfected with V 2 R. Increased cell Cl Ϫ conductance after agonist-induced cAMP elevation was assayed using a plate reader from cell fluorescence after solution I Ϫ addition. The ZЈ factor for the assay was ϳ0.7 with the V 2 R agonist [deamino-Cys1, Val4, D-Arg8]-vasopressin (1 nM) as positive control. Primary screening of 50,000 small molecules yielded a novel, 5-aryl-4-benzoyl-3-hydroxy-1-(2-arylethyl)-2H-pyrrol-2-one class of V 2 R antagonists that are unrelated structurally to known V 2 R antagonists. The most potent compound, V 2 R inh -02, which was identified by screening 35 structural analogs, competitively inhibited V 2 R-induced cAMP elevation with K i value of ϳ70 nM and fully displaced radiolabeled vasopressin in binding experiments. V 2 R inh -02 did not inhibit forskolin or ␤ 2 -adrenergic receptor-induced cAMP production and was more than 50 times more potent for V 2 R than for V 1a R. The favorable in vitro properties of the pyrrol-2-one antagonists suggests their potential usefulness in aquaretic applications. The CFTR-linked cAMP assay developed here is applicable for efficient, high-throughput identification of modulators of cAMP-coupled GPCRs

    Affinity-matured ‘aquaporumab’ anti-aquaporin-4 antibody for therapy of seropositive neuromyelitis optica spectrum disorders

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    Pathogenesis in seropositive neuromyelitis optica spectrum disorders (herein called NMO) involves binding of IgG1 autoantibodies to aquaporin-4 (AQP4) on astrocytes in the central nervous system, which initiates complement and cellular injury. We previously developed an antibody blocking approach for potential therapy of NMO in which an engineered, monoclonal, anti-AQP4 antibody lacking cytotoxicity effector functions (called aquaporumab) blocked binding of NMO autoantibodies to astrocyte AQP4 (Tradtrantip et al. Ann. Neurol. 71, 314-322, 2012). Here, a high-affinity aquaporumab, which was generated by affinity maturation using saturation mutagenesis, was shown to block cellular injury caused by NMO patient sera. Anti-AQP4 antibody rAb-53, a fully human antibody with effector function neutralizing Fc mutations L234A/L235A and affinity-enhancing Fab mutations Y50R/S56R, called AQmabAM, bound to AQP4 in cell cultures with Kd ~ 18 ng/ml (~0.12 nM), ~8-fold greater affinity than the original antibody. AQmabAM, but without L234A/L235A Fc mutations, produced complement-dependent cytotoxicity (CDC) with EC50 ~ 82 ng/ml. AQmabAM prevented CDC produced by sera from eight NMO patients with IC50 ranging from 40 to 80 ng/ml, and similarly prevented antibody-dependent cellular cytotoxicity (ADCC). Mechanistic studies demonstrated that AQmabAM blocked binding of serum NMO autoantibodies to AQP4. AQmabAM offers a targeted, non-immunosuppressive approach for therapy of seropositive NMO. Autoantibody blocking may be a useful therapeutic strategy for other autoimmune diseases as well

    CD55 upregulation in astrocytes by statins as potential therapy for AQP4-IgG seropositive neuromyelitis optica.

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    BackgroundNeuromyelitis optica spectrum disorder (herein called NMO) is an inflammatory demyelinating disease that can be initiated by binding of immunoglobulin G autoantibodies (AQP4-IgG) to aquaporin-4 on astrocytes, causing complement-dependent cytotoxicity (CDC) and downstream inflammation. The increased NMO pathology in rodents deficient in complement regulator protein CD59 following passive transfer of AQP4-IgG has suggested the potential therapeutic utility of increasing the expression of complement regulator proteins.MethodsA cell-based ELISA was developed to screen for pharmacological upregulators of endogenous CD55 and CD59 in a human astrocyte cell line. A statin identified from the screen was characterized in cell culture models and rodents for its action on complement regulator protein expression and its efficacy in models of seropositive NMO.ResultsScreening of ~ 11,500 approved and investigational drugs and nutraceuticals identified transcriptional upregulators of CD55 but not of CD59. Several statins, including atorvastatin, simvastatin, lovastatin, and fluvastatin, increased CD55 protein expression in astrocytes, including primary cultures, by three- to four-fold at 24 h, conferring significant protection against AQP4-IgG-induced CDC. Mechanistic studies revealed that CD55 upregulation involves inhibition of the geranylgeranyl transferase pathway rather than inhibition of cholesterol biosynthesis. Oral atorvastatin at 10-20 mg/kg/day for 3 days strongly increased CD55 immunofluorescence in mouse brain and spinal cord and reduced NMO pathology following intracerebral AQP4-IgG injection.ConclusionAtorvastatin or other statins may thus have therapeutic benefit in AQP4-IgG seropositive NMO by increasing CD55 expression, in addition to their previously described anti-inflammatory and immunomodulatory actions
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