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Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune inflammatory disease of the central nervous system. Most NMOSD patients are seropositive for immunoglobulin G (IgG) autoantibodies against astrocyte water channel aquaporin-4 (AQP4), called AQP4-IgG. AQP4-IgG binding to aquaporin-4 causes complement-dependent cytotoxicity (CDC), leading to inflammation and demyelination. Here, CDC was measured in AQP4-expressing cells exposed to human complement and heat-inactivated sera from 108 AQP4-IgG seropositive NMOSD subjects and 25 non-NMOSD controls. AQP4-IgG positive sera produced a wide range of CDC, with 50% maximum cytotoxicity produced by as low as 0.2% serum concentration. Unexpectedly, 58 samples produced no cytotoxicity, and of those, four sera were cytoprotective against cytotoxic AQP4-IgG. Cytoprotection was found against different cytotoxic monoclonal AQP4-IgGs and NMOSD patient sera, and in primary astrocyte cultures. Mechanistic studies revealed that the protective factor is an IgG antibody that did not inhibit complement directly, but interfered with binding of cytotoxic AQP4-IgG to AQP4 and consequent C1q binding and complement activation. Further studies suggested that non-pathogenic AQP4-IgG, perhaps with altered glycosylation, may contribute to reduced or ineffectual binding of cytotoxic AQP4-IgG, as well as reduced cell-surface AQP4. The presence of natural cytoprotective antibodies in AQP4-IgG seropositive sera reveals an added level of complexity in NMOSD disease pathogenesis, and suggests the potential therapeutic utility of 'convalescent' serum or engineered protective antibody to interfere with pathogenic antibody in AQP4-IgG seropositive NMOSD., (© 2021. The Author(s).)

Introduction: Neuromyelitis optica spectrum disorder (NMOSD) is characterized by central nervous system inflammation and demyelination. In AQP4-IgG seropositive NMOSD, circulating immunoglobulin G (IgG) autoantibodies against astrocyte water channel aquaporin-4 (AQP4) cause tissue injury. Compelling evidence supports a pathogenic role for complement activation following AQP4-IgG binding to AQP4. Clinical studies supported the approval of eculizumab, an inhibitor of C5 cleavage, in AQP4-IgG seropositive NMOSD., Areas Covered: This review covers in vitro, animal models, and human evidence for complement-dependent and complement-independent tissue injury in AQP4-IgG seropositive NMOSD. Complement targets are discussed, including complement proteins, regulators and anaphylatoxin receptors, and corresponding drug candidates., Expert Opinion: Though preclinical data support a central pathogenic role of complement activation in AQP4-IgG seropositive NMOSD, they do not resolve the relative contributions of complement-dependent vs. complement-independent disease mechanisms such as antibody-dependent cellular cytotoxicity, T cell effector mechanisms, and direct AQP4-IgG-induced cellular injury. The best evidence that complement-dependent mechanisms predominate in AQP4-IgG seropositive NMOSD comes from eculizumab clinical data. Various drug candidates targeting distinct complement effector mechanisms may offer improved safety and efficacy. However, notwithstanding the demonstrated efficacy of complement inhibition in AQP4-IgG seropositive NMOSD, the ultimate niche for complement inhibition is not clear given multiple drug options with alternative mechanisms of action. Abbreviations: AAV2, Adeno-associated virus 2; ADCC, antibody-dependent cellular cytotoxicity; ANCA, antineutrophilic cytoplasmic autoantibody; AQP4, aquaporin-4; AQP4-IgG, AQP4-immunoglobulin G; C1-INH, C1-esterase inhibitor; C3aR, C3a receptor; C4BP, C4 binding protein; C5aR, C5a receptor; CDC, complement-dependent cytotoxicity; CFHR1, complement factor H related 1; CNS, central nervous system; EAE, experimental autoimmune encephalomyelitis; EndoS, endoglycosidase S; FHL-1, factor-H-like protein 1; GFAP, glial fibrillary acidic protein; Iba-1, ionized calcium-binding adaptor protein-1; IgG, immunoglobulin G; IVIG, intravenous human immunoglobulin G; MAC, membrane attack complex; MBL, maltose-binding lectin; MBP, myelin basic protein; MOG, myelin oligodendrocyte glycoprotein; NK cell, natural killer cell; NMOSD, neuromyelitis optica spectrum disorder; OAP, orthogonal arrays of particles; PNH, paroxysmal nocturnal hemoglobinuria.

Introduction : Neuromyelitis optica spectrum disorder (NMOSD) is an inflammatory demyelinating disease of the central nervous system affecting primarily the spinal cord and optic nerves. Most NMOSD patients are seropositive for immunoglobulin G autoantibodies against astrocyte water channel aquaporin-4, called AQP4-IgG, which cause astrocyte injury leading to demyelination and neurological impairment. Current therapy for AQP4-IgG seropositive NMOSD includes immunosuppression, B cell depletion, and plasma exchange. Newer therapies target complement, CD19 and IL-6 receptors. Areas covered : This review covers early-stage pre-clinical therapeutic approaches for seropositive NMOSD. Targets include pathogenic AQP4-IgG autoantibodies and their binding to AQP4, complement-dependent and cell-mediated cytotoxicity, blood-brain barrier, remyelination and immune effector and regulatory cells, with treatment modalities including small molecules, biologics, and cells. Expert opinion : Though newer NMOSD therapies appear to have increased efficacy in reducing relapse rate and neurological deficit, increasingly targeted therapies could benefit NMOSD patients with ongoing relapses and could potentially be superior in efficacy and safety. Of the various early-stage therapeutic approaches, IgG inactivating enzymes, aquaporumab blocking antibodies, drugs targeting early components of the classical complement system, complement regulator-targeted drugs, and Fc-based multimers are of interest. Curative strategies, perhaps involving AQP4 tolerization, remain intriguing future possibilities.

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 AQmab AM , bound to AQP4 in cell cultures with K d ~ 18 ng/ml (~0.12 nM), ~8-fold greater affinity than the original antibody. AQmab AM , but without L234A/L235A Fc mutations, produced complement-dependent cytotoxicity (CDC) with EC 50 ~ 82 ng/ml. AQmab AM prevented CDC produced by sera from eight NMO patients with IC 50 ranging from 40 to 80 ng/ml, and similarly prevented antibody-dependent cellular cytotoxicity (ADCC). Mechanistic studies demonstrated that AQmab AM blocked binding of serum NMO autoantibodies to AQP4. AQmab AM 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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Neuromyelitis 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., Methods: A 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., Results: Screening 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., Conclusion: Atorvastatin 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.

Intravenous human immunoglobulin G (IVIG) may have therapeutic benefit in neuromyelitis optica spectrum disorders (herein called NMO), in part because of the anti-inflammatory properties of the IgG Fc region. Here, we evaluated recombinant Fc hexamers consisting of the IgM μ-tailpiece fused with the Fc region of human IgG1. In vitro, the Fc hexamers prevented cytotoxicity in aquaporin-4 (AQP4) expressing cells and in rat spinal cord slice cultures exposed to NMO anti-AQP4 autoantibody (AQP4-IgG) and complement, with >500-fold greater potency than IVIG or monomeric Fc fragments. Fc hexamers at low concentration also prevented antibody-dependent cellular cytotoxicity produced by AQP4-IgG and natural killer cells. Serum from rats administered a single intravenous dose of Fc hexamers at 50 mg/kg taken at 8 h did not produce complement-dependent cytotoxicity when added to AQP4-IgG-treated AQP4-expressing cell cultures. In an experimental rat model of NMO produced by intracerebral injection of AQP4-IgG, Fc hexamers at 50 mg/kg administered before and at 12 h after AQP4-IgG fully prevented astrocyte injury, complement activation, inflammation and demyelination. These results support the potential therapeutic utility of recombinant IgG1 Fc hexamers in AQP4-IgG seropositive NMO., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Introduction: Aquaporin-4 (AQP4) is a water transporting protein expressed at the plasma membrane of astrocytes throughout the central nervous system (CNS). Analysis of AQP4 knockout mice has suggested its broad involvement in brain water balance, neuroexcitation, glial scarring, neuroinflammation, and even neurodegenerative and neuropsychiatric disorders. Broad clinical utility of AQP4 modulators has been speculated. Area covered: This review covers the biology of AQP4, evidence for its roles in normal CNS function and neurological disorders, and progress in AQP4 drug discovery. Expert opinion: Critical examination of available data reduces the lengthy potential applications list to AQP4 inhibitors for early therapy of ischemic stroke and perhaps for reduction of glial scarring following CNS injury. Major challenges in identification and clinical development of AQP4 inhibitors include the apparent poor druggability of AQPs, the many homologous AQP isoforms with broad tissue distribution and functions, technical issues with water transport assays, predicted undesired CNS and non-CNS actions, and the need for high blood-brain barrier permeation. To date, despite considerable effort, validated small-molecule AQP4 inhibitors have not been advanced. However, a biologic ('aquaporumab') is in development for neuromyelitis optica, an autoimmune inflammatory demyelinating disease where CNS pathology is initiated by binding of anti-AQP4 autoantibodies to astrocyte AQP4.

Neuromyelitis optica spectrum disorder (herein called NMO) is an autoimmune inflammatory disease of the central nervous system in which immunoglobulin G antibodies against astrocyte water channel aquaporin-4 (AQP4-IgG) cause demyelination and neurological deficit. Injury to oligodendrocytes, which do not express AQP4, links the initiating pathogenic event of AQP4-IgG binding to astrocyte AQP4 to demyelination. Here, we report evidence for a complement 'bystander mechanism' to account for early oligodendrocyte injury in NMO in which activated, soluble complement proteins following AQP4-IgG binding to astrocyte AQP4 result in deposition of the complement membrane attack complex (MAC) on nearby oligodendrocytes. Primary cocultures of rat astrocytes and mature oligodendrocytes exposed to AQP4-IgG and complement showed early death of oligodendrocytes in close contact with astrocytes, which was not seen in pure oligodendrocyte cultures, in cocultures exposed to AQP4-IgG and C6-depleted serum, or when astrocytes were damaged by a complement-independent mechanism. Astrocyte-oligodendrocyte cocultures exposed to AQP4-IgG and complement showed prominent MAC deposition on oligodendrocytes in contact with astrocytes, whereas C1q, the initiating protein in the classical complement pathway, and C3d, a component of the alternative complement pathway, were deposited only on astrocytes. Early oligodendrocyte injury with MAC deposition was also found in rat brain following intracerebral injection of AQP4-IgG, complement and a fixable dead-cell stain. These results support a novel complement bystander mechanism for early oligodendrocyte injury and demyelination in NMO.

Secretory diarrheas caused by bacterial enterotoxins, including cholera and traveler's diarrhea, remain a major global health problem. Inappropriate activation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel occurs in these diarrheas. We previously reported that the benzopyrimido-pyrrolo-oxazinedione (R)-BPO-27 inhibits CFTR chloride conductance with low-nanomolar potency. Here, we demonstrate using experimental mouse models and human enterocyte cultures the potential utility of (R)-BPO-27 for treatment of secretory diarrheas caused by cholera and Escherichia coli enterotoxins. (R)-BPO-27 fully blocked CFTR chloride conductance in epithelial cell cultures and intestine after cAMP agonists, cholera toxin, or heat-stable enterotoxin of E. coli (STa toxin), with IC 50 down to ∼5 nM. (R)-BPO-27 prevented cholera toxin and STa toxin-induced fluid accumulation in small intestinal loops, with IC 50 down to 0.1 mg/kg. (R)-BPO-27 did not impair intestinal fluid absorption or inhibit other major intestinal transporters. Pharmacokinetics in mice showed >90% oral bioavailability with sustained therapeutic serum levels for >4 h without the significant toxicity seen with 7-d administration at 5 mg/kg/d. As evidence to support efficacy in human diarrheas, (R)-BPO-27 blocked fluid secretion in primary cultures of enteroids from human small intestine and anion current in enteroid monolayers. These studies support the potential utility of (R)-BPO-27 for therapy of CFTR-mediated secretory diarrheas.-Cil, O., Phuan, P.-W., Gillespie, A. M., Lee, S., Tradtrantip, L., Yin, J., Tse, M., Zachos, N. C., Lin, R., Donowitz, M., Verkman, A. S. Benzopyrimido-pyrrolo-oxazine-dione CFTR inhibitor (R)-BPO-27 for antisecretory therapy of diarrheas caused by bacterial enterotoxins., (© FASEB.)

The aquaporins (AQPs) are a family of water-transporting proteins that are broadly expressed in mammalian cells. Two AQPs in the central nervous system, AQP1 and AQP4, might play a role in hydrocephalus and are thus potential drug targets. AQP1 is expressed in the ventricular-facing membrane of choroid plexus epithelial cells, where it facilitates the secretion of cerebrospinal fluid (CSF). AQP4 is expressed in astrocyte foot processes and ependymal cells lining ventricles, where it appears to facilitate the transport of excess water out of the brain. Altered expression of these AQPs in experimental animal models of hydrocephalus and limited human specimens suggests their involvement in the pathophysiology of hydrocephalus, as do data in knockout mice demonstrating a protective effect of AQP1 deletion and a deleterious effect of AQP4 deletion in hydrocephalus. Though significant questions remain, including the precise contribution of AQP1 to CSF secretion in humans and the mechanisms by which AQP4 facilitates clearance of excess brain water, AQP1 and AQP4 have been proposed as potential drug targets to reduce ventricular enlargement in hydrocephalus., (© 2016 S. Karger AG, Basel.)

Drugs targeting aquaporins have broad potential clinical applications, including cancer, obesity, edema, glaucoma, skin diseases and others. The astrocyte water channel aquaporin-4 is a particularly compelling target because of its role of brain water movement, neuroexcitation and glia scarring, and because it is the target of pathogenic autoantibodies in the neuroinflammatory demyelinating disease neuromyelitis optica . There has been considerable interest in the identification of small molecule inhibitors of aquaporins, with various candidates emerging from testing of known ion transport inhibitors, as well as compound screening and computational chemistry. However, in general, the activity of reported aquaporin inhibitors has not been confirmed on retesting, which may be due to technical problems in water transport assays used in the original identification studies, and the challenges in modulating the activity of small, compact, pore-containing membrane proteins. We review here the state of the field of aquaporin-modulating small molecules and biologics, and the challenges and opportunities in moving forward.

Neuromyelitis optica (NMO) is an autoimmune demyelinating disease of the central nervous system in which binding of anti-aquaporin-4 (AQP4) autoantibodies (NMO-IgG) to astrocytes causes complement-dependent cytotoxicity (CDC) and inflammation resulting in oligodendrocyte and neuronal injury. There is compelling evidence for a central role of complement in NMO pathogenesis. Here, we evaluated the potential of C1-esterase inhibitor (C1-inh) for complement-targeted therapy of NMO. C1-inh is an anti-inflammatory plasma protein with serine protease inhibition activity that has a broad range of biological activities on the contact (kallikrein), coagulation, fibrinolytic and complement systems. C1-inh is approved for therapy of hereditary angioedema (HAE) and has been studied in a small safety trial in acute NMO relapses (NCT 01759602). In vitro assays of NMO-IgG-dependent CDC showed C1-inh inhibition of human and rat complement, but with predicted minimal complement inhibition activity at a dose of 2000 units in humans. Inhibition of complement by C1-inh was potentiated by ∼10-fold by polysulfated macromolecules including heparin and dextran sulfate. In rats, intravenous C1-inh at a dose 30-fold greater than that approved to treat HAE inhibited serum complement activity by <5%, even when supplemented with heparin. Also, high-dose C1-inh did not reduce pathology in a rat model of NMO produced by intracerebral injection of NMO-IgG. Therefore, although C1r and C1s are targets of C1-inh, our in vitro data with human serum and in vivo data in rats suggest that the complement inhibition activity of C1-inh in serum is too low to confer clinical benefit in NMO.

Screening of herbal remedies for Cl(-) channel inhibition identified Krisanaklan, a herbal extract used in Thailand for treatment of diarrhea, as an effective antidiarrheal in mouse models of secretory diarrheas with inhibition activity against three Cl(-) channel targets. Krisanaklan fully inhibited cholera toxin-induced intestinal fluid secretion in a closed-loop mouse model with ∼50% inhibition at a 1 ∶ 50 dilution of the extract. Orally administered Krisanaklan (5 µL/g) prevented rotavirus-induced diarrhea in neonatal mice. Short-circuit current measurements showed full inhibition of cAMP and Ca(2+) agonist-induced Cl(-) conductance in human colonic epithelial T84 cells, with ∼ 50% inhibition at a 1 ∶ 5,000 dilution of the extract. Krisanaklan also strongly inhibited intestinal smooth muscle contraction in an ex vivo preparation. Together with measurements using specific inhibitors, we conclude that the antidiarrheal actions of Krisanaklan include inhibition of luminal CFTR and Ca(2+)-activated Cl(-) channels in enterocytes. HPLC fractionation indicated that the three Cl(-) inhibition actions of Krisanaklan are produced by different components in the herbal extract. Testing of individual herbs comprising Krisanaklan indicated that agarwood and clove extracts as primarily responsible for Cl(-) channel inhibition. The low cost, broad antidiarrheal efficacy, and defined cellular mechanisms of Krisanaklan suggests its potential application for antisecretory therapy of cholera and other enterotoxin-mediated secretory diarrheas in developing countries.

Diarrheal diseases constitute a significant global health burden and are a major cause of childhood mortality and morbidity. Treatment of diarrheal disease has centered on the replacement of fluid and electrolyte losses using oral rehydration solutions. Although oral rehydration solutions have been highly successful, significant mortality and morbidity due to diarrheal disease remains. Secretory diarrheas, such as those caused by bacterial and viral enterotoxins, result from activation of cyclic nucleotide and/or Ca(2+) signaling pathways in intestinal epithelial cells, enterocytes, which increase the permeability of Cl(-) channels at the lumen-facing membrane. Additionally, there is often a parallel reduction in intestinal Na(+) absorption. Inhibition of enterocyte Cl(-) channels, including the cystic fibrosis transmembrane conductance regulator and Ca(2+)-activated Cl(-) channels, represents an attractive strategy for antisecretory drug therapy. High-throughput screening of synthetic small-molecule collections has identified several classes of Cl(-) channel inhibitors that show efficacy in animal models of diarrhea but remain to be tested clinically. In addition, several natural product extracts with Cl(-) channel inhibition activity have shown efficacy in diarrhea models. However, a number of challenges remain to translate the promising bench science into clinically useful therapeutics, including efficiently targeting orally administered drugs to enterocytes during diarrhea, funding development costs, and carrying out informative clinical trials. Nonetheless, Cl(-) channel inhibitors may prove to be effective adjunctive therapy in a broad spectrum of clinical diarrheas, including acute infectious and drug-related diarrheas, short bowel syndrome, and congenital enteropathies., (Copyright © 2014. Published by Elsevier Inc.)

The water channel aquaporin-4 (AQP4) is the target of the immunoglobulin G autoantibody (AQP4-IgG) in neuromyelitis optica (NMO). AQP4 is expressed in foot processes of astrocytes throughout the central nervous system, as well as in skeletal muscle and epithelial cells in kidney, lung and gastrointestinal organs. Phenotype analysis of AQP4 knockout mice indicates the involvement of AQP4 in water movement into and out of the brain, astrocyte migration, glial scar formation and neuroexcitatory phenomena. AQP4 monomers form tetramers in membranes, which further aggregate to form supramolecular assemblies called orthogonal arrays of particles. AQP4-IgG is pathogenic in NMO by a mechanism involving complement- and cell-mediated astrocyte cytotoxicity, which produces an inflammatory response with oligodendrocyte injury and demyelination. AQP4 orthogonal arrays are crucial in NMO pathogenesis, as they increase AQP4-IgG binding to AQP4 and greatly enhance complement-dependent cytotoxicity. Novel NMO therapeutics are under development that target AQP4-IgG or AQP4, including aquaporumab monoclonal antibodies and small molecules that block AQP4-IgG binding to AQP4, and enzymatic inactivation strategies to neutralize AQP4-IgG pathogenicity., (© 2013 International Society of Neuropathology.)

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 antibody-dependent 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(ab')(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(ab')(2) generated by IdeS cleavage. NMO-F(ab')(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. IgG-selective cleavage by IdeS thus neutralizes NMO-IgG pathogenicity, and yields therapeutic F(ab')(2) and Fc fragments. IdeS treatment, by therapeutic apheresis or direct administration, may be beneficial in NMO.

Neuromyelitis optica (NMO) is an autoimmune disorder with inflammatory demyelinating lesions in the central nervous system, particularly in the spinal cord and optic nerve. NMO pathogenesis is thought to involve binding of anti-aquaporin-4 (AQP4) autoantibodies to astrocytes, which causes complement-dependent cytotoxicity (CDC) and downstream inflammation leading to oligodendrocyte and neuronal injury. Vasculocentric deposition of activated complement is a prominent feature of NMO pathology. Here, we show that a neutralizing monoclonal antibody against the C1q protein in the classical complement pathway prevents AQP4 autoantibody-dependent CDC in cell cultures and NMO lesions in ex vivo spinal cord slice cultures and in mice. A monoclonal antibody against human C1q with 11 nM binding affinity prevented CDC caused by NMO patient serum in AQP4-transfected cells and primary astrocyte cultures, and prevented complement-dependent cell-mediated cytotoxicity (CDCC) produced by natural killer cells. The anti-C1q antibody prevented astrocyte damage and demyelination in mouse spinal cord slice cultures exposed to AQP4 autoantibody and human complement. In a mouse model of NMO produced by intracerebral injection of AQP4 autoantibody and human complement, the inflammatory demyelinating lesions were greatly reduced by intracerebral administration of the anti-C1q antibody. These results provide proof-of-concept for C1q-targeted monoclonal antibody therapy in NMO. Targeting of C1q inhibits the classical complement pathway directly and causes secondary inhibition of CDCC and the alternative complement pathway. As C1q-targeted therapy leaves the lectin complement activation pathway largely intact, its side-effect profile is predicted to differ from that of therapies targeting downstream complement proteins.

We previously reported benzopyrimido-pyrrolo-oxazinedione (BPO) inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and showed their efficacy in a model of polycystic kidney disease. Here, we separated the enantiomers of lead compound BPO-27, ( 1 ), which contains a single chiral center, and determined their absolute configuration, activity and metabolic stability. Following separation by chiral supercritical fluid chromatography, the R enantiomer, as determined by x-ray crystallography, inhibited CFTR chloride conductance with IC 50 ~ 4 nM, while S enantiomer was inactive. In vitro metabolic stability in hepatic microsomes showed both enantiomers as stable, with <5 % metabolism in 4 h. Following bolus interperitoneal administration in mice, serum ( R )- 1 decayed with t 1/2 ~ 1.6 h and gave sustained therapeutic concentrations in kidney.

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a cAMP-regulated Cl- channel whose major function is to facilitate epithelial fluid secretion. Loss-of-function mutations in CFTR cause the genetic disease cystic fibrosis. CFTR is required for transepithelial fluid transport in certain secretory diarrheas, such as cholera, and for cyst expansion in autosomal dominant polycystic kidney disease. High-throughput screening has yielded CFTR inhibitors of the thiazolidinone, glycine hydrazide and quinoxalinedione chemical classes. The glycine hydrazides target the extracellular CFTR pore, whereas the thiazolidinones and quinoxalinediones act at the cytoplasmic surface. These inhibitors have been widely used in cystic fibrosis research to study CFTR function at the cell and organ levels. The most potent CFTR inhibitor has IC50 of approximately 4 nM. Studies in animal models support the development of CFTR inhibitors for antisecretory therapy of enterotoxin-mediated diarrheas and polycystic kidney disease.

Objective: Neuromyelitis optica (NMO) is caused by binding of pathogenic autoantibodies (NMO-immunoglobulin G [IgG]) to aquaporin-4 (AQP4) on astrocytes, which initiates complement-dependent cytotoxicity (CDC) and inflammation. We recently introduced mutated antibody (aquaporumab) and small-molecule blocker strategies for therapy of NMO, based on prevention of NMO-IgG binding to AQP4. Here, we investigated an alternative strategy involving neutralization of NMO-IgG effector function by selective IgG heavy-chain deglycosylation with bacteria-derived endoglycosidase S (EndoS)., Methods: Cytotoxicity and NMO pathology were measured in cell and spinal cord slice cultures, and in mice exposed to control or EndoS-treated NMO-IgG., Results: EndoS treatment of NMO patient serum reduced by >95% CDC and antibody-dependent cell-mediated cytotoxicity, without impairment of NMO-IgG binding to AQP4. Cytotoxicity was also prevented by addition of EndoS after NMO-IgG binding to AQP4. The EndoS-treated, nonpathogenic NMO-IgG competitively displaced pathogenic NMO-IgG bound to AQP4, and prevented NMO pathology in spinal cord slice culture and mouse models of NMO., Interpretation: EndoS deglycosylation converts pathogenic NMO-IgG autoantibodies into therapeutic blocking antibodies. EndoS treatment of blood may be beneficial in NMO, and may be accomplished, for example, by therapeutic apheresis using surface-immobilized EndoS., (Copyright © 2012 American Neurological Association.)

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system caused by binding of anti-aquaporin-4 (AQP4) autoantibodies (NMO-IgG) to AQP4 on astrocytes. A screen was developed to identify inhibitors of NMO-IgG-dependent, complement-dependent cytotoxicity. Screening of 50,000 synthetic small molecules was done using CHO cells expressing human AQP4 and a human NMO recombinant monoclonal antibody (rAb-53). The screen yielded pyrano[2,3-c]pyrazoles that blocked rAb-53 binding to AQP4 and prevented cytotoxicity in cell culture and spinal cord slice models of NMO. Structure-activity analysis of 82 analogs yielded a blocker with IC(50) ∼ 6 μm. Analysis of the blocker mechanism indicated idiotype specificity, as (i) pyrano[2,3-c]pyrazoles did not prevent AQP4 binding or cytotoxicity of other NMO-IgGs, and (ii) surface plasmon resonance showed specific rAb-53 binding. Antibody structure modeling and docking suggested a putative binding site near the complementarity-determining regions. Small molecules with idiotype-specific antibody targeting may be useful as research tools and therapeutics.

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of spinal cord and optic nerve caused by pathogenic autoantibodies (NMO-IgG) against astrocyte aquaporin-4 (AQP4). We developed a high-throughput screen to identify blockers of NMO-IgG binding to human AQP4 using a human recombinant monoclonal NMO-IgG and transfected Fisher rat thyroid cells stably expressing human M23-AQP4. Screening of ∼60,000 compounds yielded the antiviral arbidol, the flavonoid tamarixetin, and several plant-derived berbamine alkaloids, each of which blocked NMO-IgG binding to AQP4 without affecting AQP4 expression, array assembly, or water permeability. The compounds inhibited NMO-IgG binding to AQP4 in NMO patient sera and blocked NMO-IgG-dependent complement- and cell-mediated cytotoxicity with IC(50) down to ∼5 μM. Docking computations identified putative sites of blocker binding at the extracellular surface of AQP4. The blockers did not affect complement-dependent cytotoxicity caused by anti-GD3 antibody binding to ganglioside GD3. The blockers reduced by >80% the severity of NMO lesions in an ex vivo spinal cord slice culture model of NMO and in mice in vivo. Our results provide proof of concept for a small-molecule blocker strategy to reduce NMO pathology. Small-molecule blockers may also be useful for other autoimmune diseases caused by binding of pathogenic autoantibodies to defined targets.

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system in which binding of pathogenic autoantibodies (NMO-IgG) to astrocyte aquaporin-4 (AQP4) causes complement-dependent cytotoxicity (CDC) and inflammation. We previously reported a wide range of binding affinities of NMO-IgGs to AQP4 in separate tetramers versus intramembrane aggregates (orthogonal arrays of particles, OAPs). We report here a second, independent mechanism by which CDC is affected by AQP4 assembly. Utilizing lactate dehydrogenase release and live/dead cell cytotoxicity assays, we found in different cell lines, and with different monoclonal and patient-derived NMO-IgGs, that CDC was greatly (>100-fold) reduced in cells expressing M1- versus M23-AQP4. Studies using a M23-AQP4 mutant containing an OAP-disrupting mutation, and in cells expressing AQP4 in different M1/M23 ratios, indicated that NMO-IgG-dependent CDC requires AQP4 OAP assembly. In contrast, antibody-dependent cell-mediated cytotoxicity produced by natural killer cells did not depend on AQP4 OAP assembly. Measurements of C1q binding and complement attack complex (C9neo) supported the conclusion that the greatly enhanced CDC by OAPs is due to efficient, multivalent binding of C1q to clustered NMO-IgG on OAPs. We conclude that AQP4 assembly in OAPs is required for CDC in NMO, establishing a new mechanism of OAP-dependent NMO pathogenesis. Disruption of AQP4 OAPs may greatly reduce NMO-IgG dependent CDC and NMO pathology.

Objective: Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system. Circulating autoantibodies (NMO-immunoglobulin [Ig]G) against astrocyte water channel aquaporin-4 (AQP4) cause complement- and cell-mediated astrocyte damage with consequent neuroinflammation and demyelination. Current NMO therapies, which have limited efficacy, include immunosuppression and plasma exchange. The objective of this study was to develop a potential new NMO therapy based on blocking of pathogenic NMO-IgG binding to its target, AQP4., Methods: We generated nonpathogenic recombinant monoclonal anti-AQP4 antibodies that selectively block NMO-IgG binding to AQP4. These antibodies comprise a tight-binding anti-AQP4 Fab and a mutated Fc that lacks functionality for complement- and cell-mediated cytotoxicity. The efficacy of the blocking antibodies was studied using cell culture, spinal cord slice, and in vivo mouse models of NMO., Results: In AQP4-expressing cell cultures, the nonpathogenic competing antibodies blocked binding of NMO-IgG in human sera, reducing to near zero complement- and cell-mediated cytotoxicity. The antibodies prevented the development of NMO lesions in an ex vivo spinal cord slice model of NMO and in an in vivo mouse model, without causing cytotoxicity., Interpretation: Our results provide proof of concept for a therapy of NMO with blocking antibodies. The broad efficacy of antibody inhibition is likely due to steric competition because of its large physical size compared to AQP4. Blocker therapy to prevent binding of pathogenic autoantibodies to their targets may be useful for treatment of other autoimmune diseases as well., (Copyright © 2011 American Neurological Association.)

The Ca(2+)-activated Cl(-) channel TMEM16A is involved in epithelial fluid secretion, smooth muscle contraction and neurosensory signaling. We identified a Thai herbal antidiarrheal formulation that inhibited TMEM16A Cl(-) conductance. C18-reversed-phase HPLC fractionation of the herbal formulation revealed >98% of TMEM16A inhibition activity in one out of approximately 20 distinct peaks. The purified, active compound was identified as eugenol (4-allyl-2-methoxyphenol), the major component of clove oil. Eugenol fully inhibited TMEM16A Cl(-) conductance with single-site IC(50)~150 µM. Eugenol inhibition of TMEM16A in interstitial cells of Cajal produced strong inhibition of intestinal contraction in mouse ileal segments. TMEM16A Cl(-) channel inhibition adds to the list of eugenol molecular targets and may account for some of its biological activities.

We previously reported the discovery of pyrimido-pyrrolo-quinoxalinedione (PPQ) inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and showed their efficacy in an organ culture model of polycystic kidney disease (PKD) (J. Med. Chem. 2009, 52, 6447-6455). Here, we report related benzopyrimido-pyrrolo-oxazinedione (BPO) CFTR inhibitors. To establish structure-activity relationships and select lead compound(s) with improved potency, metabolic stability, and aqueous solubility compared to the most potent prior compound 8 (PPQ-102, IC(50) ∼ 90 nM), we synthesized 16 PPQ analogues and 11 BPO analogues. The analogues were efficiently synthesized in 5-6 steps and 11-61% overall yield. Modification of 8 by bromine substitution at the 5-position of the furan ring, replacement of the secondary amine with an ether bridge, and carboxylation, gave 6-(5-bromofuran-2-yl)-7,9-dimethyl-8,10-dioxo-11-phenyl-7,8,9,10-tetrahydro-6H-benzo[b]pyrimido [4',5':3,4]pyrrolo [1,2-d][1,4]oxazine-2-carboxylic acid 42 (BPO-27), which fully inhibited CFTR with IC(50) ∼ 8 nM and, compared to 8, had >10-fold greater metabolic stability and much greater polarity/aqueous solubility. In an embryonic kidney culture model of PKD, 42 prevented cyst growth with IC(50) ∼ 100 nM. Benzopyrimido-pyrrolo-oxazinediones such as 42 are potential development candidates for antisecretory therapy of PKD.

Aquaporin-4 (AQP4) is a water-selective transporter expressed in astrocytes throughout the central nervous system, as well as in kidney, lung, stomach and skeletal muscle. The two AQP4 isoforms produced by alternative spicing, M1 and M23 AQP4, form heterotetramers that assemble in cell plasma membranes in supramolecular structures called orthogonal arrays of particles (OAPs). Phenotype analysis of AQP4-null mice indicates the involvement of AQP4 in brain and spinal cord water balance, astrocyte migration, neural signal transduction and neuroinflammation. AQP4-null mice manifest reduced brain swelling in cytotoxic cerebral edema, but increased brain swelling in vasogenic edema and hydrocephalus. AQP4 deficiency also increases seizure duration, impairs glial scarring, and reduces the severity of autoimmune neuroinflammation. Each of these phenotypes is likely explicable on the basis of reduced astrocyte water permeability in AQP4 deficiency. AQP4 is also involved in the neuroinflammatory demyelinating disease neuromyelitis optica (NMO), where autoantibodies (NMO-IgG) targeting AQP4 produce astrocyte damage and inflammation. Mice administered NMO-IgG and human complement by intracerebral injection develop characteristic NMO lesions with neuroinflammation, demyelination, perivascular complement deposition and loss of glial fibrillary acidic protein and AQP4 immunoreactivity. Our findings suggest the potential utility of AQP4-based therapeutics, including small-molecule modulators of AQP4 water transport function for therapy of brain swelling, injury and epilepsy, as well as small-molecule or monoclonal antibody blockers of NMO-IgG binding to AQP4 for therapy of NMO.

Crofelemer, a purified proanthocyanidin oligomer extracted from the bark latex of Croton lechleri, is in clinical trials for secretory diarrheas of various etiologies. We investigated the antisecretory mechanism of crofelemer by determining its effect on the major apical membrane transport and signaling processes involved in intestinal fluid transport. Using cell lines and measurement procedures to isolate the effects on individual membrane transport proteins, crofelemer at 50 microM had little or no effect on the activity of epithelial Na(+) or K(+) channels or on cAMP or calcium signaling. Crofelemer inhibited the cystic fibrosis transmembrane regulator (CFTR) Cl(-) channel with maximum inhibition of approximately 60% and an IC(50) approximately 7 microM. Crofelemer action at an extracellular site on CFTR produced voltage-independent block with stabilization of the channel closed state. Crofelemer did not affect the potency of glycine hydrazide or thiazolidinone CFTR inhibitors. Crofelemer action resisted washout, with <50% reversal of CFTR inhibition after 4 h. Crofelemer was also found to strongly inhibit the intestinal calcium-activated Cl(-) channel TMEM16A by a voltage-independent inhibition mechanism with maximum inhibition >90% and IC(50) approximately 6.5 microM. The dual inhibitory action of crofelemer on two structurally unrelated prosecretory intestinal Cl(-) channels may account for its intestinal antisecretory activity.

Inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are predicted to slow cyst enlargement in polycystic kidney disease and reduce intestinal fluid loss in secretory diarrheas. Screening of approximately 110000 small synthetic and natural compounds for inhibition of halide influx in CFTR-expressing epithelial cells yielded a new class of pyrimido-pyrrolo-quinoxalinedione (PPQ) CFTR inhibitors. Testing of 347 analogues established structure-activity relationships. The most potent compound, 7,9-dimethyl-11-phenyl-6-(5-methylfuran-2-yl)-5,6-dihydro-pyrimido[4',5'-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione, PPQ-102, completely inhibited CFTR chloride current with IC(50) approximately 90 nM. The PPQs, unlike prior CFTR inhibitors, are uncharged at physiological pH, and therefore not subject to membrane potential-dependent cellular partitioning or block efficiency. Patch-clamp analysis confirmed voltage-independent CFTR inhibition by PPQ-102 and showed stabilization of the channel closed state. PPQ-102 prevented cyst expansion and reduced the size of preformed cysts in a neonatal kidney organ culture model of polycystic kidney disease. PPQ-102 is the most potent CFTR inhibitor identified to date.

We carried out a "pathway" screen of 50,000 small molecules to identify novel modulators of cAMP signaling. One class of compounds, the 2-(acylamino)-3-thiophenecarboxylates, strongly suppressed cAMP and cGMP in multiple cell lines in response to different agonists acting on G-protein-coupled receptors, adenylyl cyclase, and guanylyl cyclase. The best compounds from structure-activity analysis of 124 analogs, including several synthesized chiral analogs, had and IC(50) of <5 microM for suppression of agonist-induced cAMP and cGMP elevation. Measurements of cAMP, cGMP, and downstream signaling in response to various activators/inhibitors suggested that the 2-(acylamino)-3-thiophenecarboxylates function as nonselective phosphodiesterase activators, although it was not determined whether their action on phosphodiesterases is direct or indirect. The 2-(acylamino)-3-thiophenecarboxylates suppressed CFTR-mediated Cl(-) current in T84 colonic cells in response to cholera and Escherichia coli (STa) toxins, and prevented intestinal fluid accumulation in a closed-loop mouse model of secretory diarrhea. They also prevented cyst growth in an in vitro renal epithelial cell model of polycystic kidney disease. The 2-(acylamino)-3-thiophenecarboxylates represent the first small-molecule cyclic nucleotide suppressors, whose potential therapeutic indications include secretory diarrheas, polycystic kidney disease, and growth inhibition of cAMP-dependent tumors.

Phloracetophenone (2,4,6-trihydroxyacetophenone, THA) is a potent choleretic in the bile fistula rat, although the mechanism is unknown. In the present study, we examined how THA enhances bile secretion. Stepwise infusions of THA (1-4 micromol/min) in the isolated perfused rat liver resulted in an immediate and dose-dependent increase in bile flow (BF), which reached saturation. The increase in BF was not associated with a change in the excretion of bile acids, suggesting that THA stimulated bile acid-independent bile flow. To further define the mechanism, the effect of THA on the excretion of sulfobromophthalein (BSP) and disulfobromophthalein (DBSP), typical multidrug resistance protein-2 (Mrp2) substrates was examined. THA inhibited the biliary excretion of both substrates. Because DBSP is excreted without conjugation to glutathione, in contrast to BSP, the findings suggest that THA might compete with DBSP and BSP metabolites at a common canalicular transport site, presumably Mrp2. THA infusions had no effect on the subcellular localization and distribution of either Mrp2 or the bile salt export pump (Bsep), nor the integrity of the tight junction. In contrast, the choleretic activity of THA was completely absent in the TR(-) rat, an animal model that lacks Mrp2, directly implicating this canalicular export pump as the mechanisms by which THA is excreted in bile. THA also partially reversed the cholestatic effects of estradiol-17beta-D-glucuronide, a process also dependent on Mrp2. In conclusion, the choleretic activity of THA and its possible metabolites is dependent on Mrp2. THA appears to stimulate BF by its osmotic effects and may attenuate the cholestatic effects of hepatotoxins undergoing biotransformation and excretion via similar pathways.

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(-) 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(-) conductance after agonist-induced cAMP elevation was assayed using a plate reader from cell fluorescence after solution I(-) addition. The Z' factor for the assay was approximately 0.7 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 approximately 70 nM and fully displaced radiolabeled vasopressin in binding experiments. V(2)R(inh)-02 did not inhibit forskolin or beta(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.

Insertion of transporter proteins into the apical canalicular membrane via vesicular transport is one of several choleretic mechanisms. Based on different choleretic activities of hydroxyacetophenone analogues including 4-mono; 2,6-di and 2,4,6-trihydroxy-acetophenone (MHA, DHA and THA), the present study aims to determine if these compounds stimulated vesicular transport in hepatocytes. Hydroxyacetophenone was continuously infused into the duodenum of the bile fistula rat. Bile flow rate was allowed to stabilize and then followed by an intraportal injection of horseradish peroxidase, a marker of the transcytotic vesicle pathway. MHA which stimulates bile acid independent flow, showed a dose-dependent increase in both the early (paracellular) and late (transcellular) peak of horseradish peroxidase excretion in bile. THA, which stimulates both bile acid dependent flow and bile acid independent flow, did not alter the pattern of horseradish peroxidase excretion into bile. However, DHA, which is more hydrophobic and increases only bile acid dependent flow, decreased the late peak. The stimulating effects of MHA on bile flow and horseradish peroxidase excretion were markedly inhibited by colchicine, suggesting that its choleretic action involves stimulation of exocytosis, as well as increase in paracellular permeability. In contrast, the lack of a stimulatory effect of THA and DHA on biliary horseradish peroxidase excretion suggested that their choleretic action is not associated with vesicular exocytosis. These results demonstrate a variable effect of hydroxyacetophenones on the transcytotic vesicular pathway reflecting different choleretic mechanisms and therapeutic potential.