Marc O. Anderson

Aquaporins: important but elusive drug targets

The aquaporins (AQPs) are a family of small, integral membrane proteins that facilitate water transport across the plasma membranes of cells in response to osmotic gradients. Data from knockout mice support the involvement of AQPs in epithelial fluid secretion, cell migration, brain oedema and adipocyte metabolism, which suggests that modulation of AQP function or expression could have therapeutic potential in oedema, cancer, obesity, brain injury, glaucoma and several other conditions. Moreover, loss-of-function mutations in human AQPs cause congenital cataracts (AQP0) and nephrogenic diabetes insipidus (AQP2), and autoantibodies against AQP4 cause the autoimmune demyelinating disease neuromyelitis optica. Although some potential AQP modulators have been identified, challenges associated with the development of better modulators include the druggability of the target and the suitability of the assay methods used to identify modulators.

Global identification of peptidase specificity by multiplex substrate profiling

We developed a simple and rapid multiplex substrate-profiling method to reveal the substrate specificity of any endo- or exopeptidase using liquid chromatography–tandem mass spectrometry sequencing. We generated a physicochemically diverse library of peptides by incorporating all combinations of neighbor and near-neighbor amino acid pairs into decapeptide sequences that are flanked by unique dipeptides at each terminus. Addition of a panel of evolutionarily diverse peptidases to a mixture of these tetradecapeptides generated information on prime and nonprime sites as well as on substrate specificity that matched or expanded upon known substrate motifs. This method biochemically confirmed the activity of the klassevirus 3C protein responsible for polypeptide processing and allowed granzyme B substrates to be ranked by enzymatic turnover efficiency using label-free quantitation of precursor-ion abundance. Additionally, the proteolytic secretions from schistosome parasitic flatworm larvae and a pancreatic cancer cell line were deconvoluted in a subtractive strategy using class-specific peptidase inhibitors.

Small-molecule inhibitors of NMO-IgG binding to aquaporin-4 reduce astrocyte cytotoxicity in neuromyelitis optica

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 IC50 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.—Tradtrantip, L., Zhang, H., Anderson, M. O., Saadoun, S., Phuan, P.‐W., Papadopoulos, M. C., Bennett, J. L., Verkman, A. S. Small‐molecule inhibitors of NMO‐IgG binding to aquaporin‐4 reduce astrocyte cytotoxicity in neuromyelitis optica. FASEB J. 26, 2197‐2208 (2012). www.fasebj.org

Triazolothienopyrimidine Inhibitors of Urea Transporter UT-B Reduce Urine Concentration

Urea transport (UT) proteins facilitate the concentration of urine by the kidney, suggesting that inhibition of these proteins could have therapeutic use as a diuretic strategy. We screened 100,000 compounds for UT-B inhibition using an optical assay based on the hypotonic lysis of acetamide-loaded mouse erythrocytes. We identified a class of triazolothienopyrimidine UT-B inhibitors; the most potent compound, UTB(inh)-14, fully and reversibly inhibited urea transport with IC(50) values of 10 nM and 25 nM for human and mouse UT-B, respectively. UTB(inh)-14 competed with urea binding at an intracellular site on the UT-B protein. UTB(inh)-14 exhibited low toxicity and high selectivity for UT-B over UT-A isoforms. After intraperitoneal administration of UTB(inh)-14 in mice to achieve predicted therapeutic concentrations in the kidney, urine osmolality after administration of 1-deamino-8-D-arginine-vasopressin was approximately 700 mosm/kg H(2)O lower in UTB(inh)-14-treated mice than vehicle-treated mice. UTB(inh)-14 also increased urine output and reduced urine osmolality in mice given free access to water. UTB(inh)-14 did not reduce urine osmolality in UT-B knockout mice. In summary, these data provide proof of concept for the potential utility of UT inhibitors to reduce urinary concentration in high-vasopressin, fluid-retaining conditions. The diuretic mechanism of UT inhibitors may complement the action of conventional diuretics, which target sodium transport.

Sustained Activation of N-Methyl-d-Aspartate Receptors in Podoctyes Leads to Oxidative Stress, Mobilization of Transient Receptor Potential Canonical 6 Channels, Nuclear Factor of Activated T Cells Activation, and Apoptotic Cell Death

Atypical N-methyl-d-aspartate (NMDA) receptors are expressed in podocytes. Sustained (≥24 h) application of 50 to 100 μM NMDA to immortalized mouse podocytes evoked a marked increase in the production of reactive oxygen species (ROS) such as H2O2. This effect of NMDA was associated with increased cell-surface expression of p47(phox), a cytosolic regulatory subunit of the NADPH oxidase NOX2. NMDA-evoked generation of ROS drove an increase in steady-state surface expression of transient receptor potential canonical (TRPC) 6 channels, which was blocked by the NMDA antagonist dizocilpine (MK-801) and by a membrane-permeable scavenger of ROS. The effect of NMDA on TRPC6 was observed using cell surface biotinylation assays and also with whole-cell recordings made under conditions designed to facilitate detection of current through TRPC6. NMDA mobilization of TRPC6 channels was blocked by concurrent treatment with the NMDA antagonist MK-801 and by a membrane-permeable scavenger of ROS. Mobilization of TRPC6 was also evoked by l-homocysteic acid. NMDA treatment also increased nuclear localization of endogenous nuclear factor of activated T cells, which could be blocked by MK-801, by scavenging ROS, by the calcineurin inhibitor cyclosporine, and by the TRPC channel inhibitor 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl]imidazole (SKF-96365). NMDA treatment also evoked robust activation of Rho but not Rac, consistent with previous studies of downstream effectors of TRPC6 activation. Exposing cells to NMDA for 24 h reduced total and cell surface expression of the podocyte markers nephrin and podocin, but there was no loss of cells. With longer NMDA exposure (72 h), we observed loss of cells associated with nuclear fragmentation and increased expression of caspase-3, caspase-6, and Bax, suggesting an apoptotic process.

Synthesis of aryl-heteroaryl ureas (AHUs) based on 4-aminoquinoline and their evaluation against the insulin-like growth factor receptor (IGF-1R)

The insulin-like growth factor receptor (IGF-1R) is a receptor tyrosine kinase (RTK) involved in all stages of the development and propagation of breast and other cancers. The inhibition of IGF-1R by small molecules remains a promising strategy to treat cancer. Herein, we explore SAR around previously characterized lead compound (1), which is an aryl-heteroaryl urea (AHU) consisting of 4-aminoquinaldine and a substituted aromatic ring system. A library of novel AHU compounds was prepared based on derivatives of the 4-aminoquinoline heterocycle (including various 2-substituted derivatives, and naphthyridines). The compounds were screened for in vitro inhibitory activity against IGF-1R, and several compounds with improved activity (3-5 microM) were identified. Furthermore, a computational docking study was performed, which identifies a fairly consistent lowest energy mode of binding for the more-active set of inhibitors in this series, while the less-active inhibitors do not adopt a consistent mode of binding.

Urea transporter proteins as targets for small-molecule diuretics

Conventional diuretics such as furosemide and thiazides target salt transporters in kidney tubules, but urea transporters (UTs) have emerged as alternative targets. UTs are a family of transmembrane channels expressed in a variety of mammalian tissues, in particular the kidney. UT knockout mice and humans with UT mutations exhibit reduced maximal urinary osmolality, demonstrating that UTs are necessary for the concentration of urine. Small-molecule screening has identified potent and selective inhibitors of UT-A, the UT protein expressed in renal tubule epithelial cells, and UT-B, the UT protein expressed in vasa recta endothelial cells. Data from UT knockout mice and from rodents administered UT inhibitors support the diuretic action of UT inhibition. The kidney-specific expression of UT-A1, together with high selectivity of the small-molecule inhibitors, means that off-target effects of such small-molecule drugs should be minimal. This Review summarizes the structure, expression and function of UTs, and looks at the evidence supporting the validity of UTs as targets for the development of salt-sparing diuretics with a unique mechanism of action. UT-targeted inhibitors may be useful alone or in combination with conventional diuretics for therapy of various oedemas and hyponatraemias, potentially including those refractory to treatment with current diuretics.

Diuresis and reduced urinary osmolality in rats produced by small-molecule UT-A-selective urea transport inhibitors

Urea transport (UT) proteins of the UT‐A class are expressed in epithelial cells in kidney tubules, where they are required for the formation of a concentrated urine by countercurrent multiplication. Here, using a recently developed high‐throughput assay to identify UT‐A inhibitors, a screen of 50,000 synthetic small molecules identified UT‐A inhibitors of aryl‐thiazole, γ‐sultambenzosulfonamide, aminocarbonitrile butene, and 4‐isoxazolamide chemical classes. Structure‐activity analysis identified compounds that inhibited UT‐A selectively by a noncompetitive mechanism with IC50 down to ~1 μM. Molecular modeling identified putative inhibitor binding sites on rat UTA. To test compound efficacy in rats, formulations and administration procedures were established to give therapeutic inhibitor concentrations in blood and urine. We found that intravenous administration of an indole thiazole or a γ‐sultambenzosulfonamide at 20 mg/kg increased urine output by 3‐5‐fold and reduced urine osmolality by ~ 2‐fold compared to vehicle control rats, even under conditions of maximum antidiuresis produced by 1‐deamino‐8‐D‐arginine vasopressin (DDAVP). The diuresis was reversible and showed urea > salt excretion. The results provide proof of concept for the diuretic action of UT‐A‐selective inhibitors. UT‐A inhibitors are first in their class salt‐sparing diuretics with potential clinical indications in volume‐overload edemas and high‐vasopressin‐associated hyponatremias.—Esteva‐Font, C., Cil, O., Phuan, P.‐W., Su, T., Lee, S., Anderson, M. O., Verkman, A. S. Diuresis and reduced urinary osmolality in rats produced by small‐molecule UT‐A‐ selective urea transport inhibitors. FASEB J. 28, 3878‐3890 (2014). www.fasebj.org

X-box binding protein 1 (XBP1s) is a critical determinant of Pseudomonas aeruginosa homoserine lactone-mediated apoptosis.

Pseudomonas aeruginosa infections are associated with high mortality rates and occur in diverse conditions including pneumonias, cystic fibrosis and neutropenia. Quorum sensing, mediated by small molecules including N-(3-oxo-dodecanoyl) homoserine lactone (C12), regulates P. aeruginosa growth and virulence. In addition, host cell recognition of C12 initiates multiple signalling responses including cell death. To gain insight into mechanisms of C12-mediated cytotoxicity, we studied the role of endoplasmic reticulum stress in host cell responses to C12. Dramatic protection against C12-mediated cell death was observed in cells that do not produce the X-box binding protein 1 transcription factor (XBP1s). The leucine zipper and transcriptional activation motifs of XBP1s were sufficient to restore C12-induced caspase activation in XBP1s-deficient cells, although this polypeptide was not transcriptionally active. The XBP1s polypeptide also regulated caspase activation in cells stimulated with N-(3-oxo-tetradecanoyl) homoserine lactone (C14), produced by Yersinia enterolitica and Burkholderia pseudomallei, and enhanced homoserine lactone-mediated caspase activation in the presence of endogenous XBP1s. In C12-tolerant cells, responses to C12 including phosphorylation of p38 MAPK and eukaryotic initiation factor 2α were conserved, suggesting that C12 cytotoxicity is not heavily dependent on these pathways. In summary, this study reveals a novel and unconventional role for XBP1s in regulating host cell cytotoxic responses to bacterial acyl homoserine lactones.

A Small-molecule Screen Yields Idiotype-specific Blockers of Neuromyelitis Optica Immunoglobulin G Binding to Aquaporin-4

Background: A high-throughput cytotoxicity-based screen was developed to identify small-molecule inhibitors of NMO-IgG-AQP4 binding. Results: Pyrano[2,3-c]pyrazoles were identified as inhibitors and were shown to bind directly to the NMO monoclonal antibody used for screening but did not interfere with other NMO-IgGs that bound to AQP4. Conclusion: Pyrano[2,3-c]pyrazoles are idiotype-specific to the NMO recombinant monoclonal antibody used for screening. Significance: Our results establish a proof of concept for an antibody-targeted small-molecule blocker strategy to prevent specific antibody-antigen binding. 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 IC50 ∼ 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.