5-Hydroxymethyl-Furfural and Structurally Related Compounds Block the Ion Conductance in Human Aquaporin-1 Channels and Slow Cancer Cell Migration and Invasion.

Aquaporin-1 (AQP1) dual water and ion channels enhance migration and invasion when upregulated in leading edges of certain classes of cancer cells. Work here identifies structurally related furan compounds as novel inhibitors of AQP1 ion channels. 5-Hydroxymethyl-2-furfural (5HMF), a component of natural medicinal honeys, and three structurally related compounds, 5-nitro-2-furoic acid (5NFA), 5-acetoxymethyl-2-furaldehyde (5AMF), and methyl-5-nitro-2-furoate (M5NF), were analyzed for effects on water and ion channel activities of human AQP1 channels expressed in Xenopus oocytes. Two-electrode voltage clamp showed dose-dependent block of the AQP1 ion current by 5HMF (IC50 0.43 mM), 5NFA (IC50 1.2 mM), and 5AMF (IC50 ∼3 mM) but no inhibition by M5NF. In silico docking predicted the active ligands interacted with glycine 165, located in loop D gating domains surrounding the intracellular vestibule of the tetrameric central pore. Water fluxes through separate intrasubunit pores were unaltered by the furan compounds (at concentrations up to 5 mM). Effects on cell migration, invasion, and cytoskeletal organization in vitro were tested in high-AQP1-expressing cancer lines, colon cancer (HT29) and AQP1-expressing breast cancer (MDA), and low-AQP1-expressing SW480. 5HMF, 5NFA, and 5AMF selectively impaired cell motility in the AQP1-enriched cell lines. In contrast, M5NF immobilized all the cancer lines by disrupting actin cytoskeleton. No reduction in cell viability was observed at doses that were effective in blocking motility. These results define furans as a new class of AQP1 ion channel inhibitors for basic research and potential lead compounds for development of therapeutic agents targeting aquaporin channel activity. SIGNIFICANCE STATEMENT: 5-Hydroxymethyl-2-furfural (5HMF), a component of natural medicinal honeys, blocks the ion conductance but not the water flux through human Aquaporin-1 (AQP1) channels and impairs AQP1-dependent cell migration and invasiveness in cancer cell lines. Analyses of 5HMT and structural analogs demonstrate a structure-activity relationship for furan compounds, supported by in silico docking modeling. This work identifies new low-cost pharmacological antagonists for AQP1 available to researchers internationally. Furans merit consideration as a new class of therapeutic agents for controlling cancer metastasis.

Combined pharmacological administration of AQP1 ion channel blocker AqB011 and water channel blocker Bacopaside II amplifies inhibition of colon cancer cell migration.

Aquaporin-1 (AQP1) has been proposed as a dual water and cation channel that when upregulated in cancers enhances cell migration rates; however, the mechanism remains unknown. Previous work identified AqB011 as an inhibitor of the gated human AQP1 cation conductance, and bacopaside II as a blocker of AQP1 water pores. In two colorectal adenocarcinoma cell lines, high levels of AQP1 transcript were confirmed in HT29, and low levels in SW480 cells, by quantitative PCR (polymerase chain reaction). Comparable differences in membrane AQP1 protein levels were demonstrated by immunofluorescence imaging. Migration rates were quantified using circular wound closure assays and live-cell tracking. AqB011 and bacopaside II, applied in combination, produced greater inhibitory effects on cell migration than did either agent alone. The high efficacy of AqB011 alone and in combination with bacopaside II in slowing HT29 cell motility correlated with abundant membrane localization of AQP1 protein. In SW480, neither agent alone was effective in blocking cell motility; however, combined application did cause inhibition of motility, consistent with low levels of membrane AQP1 expression. Bacopaside alone or combined with AqB011 also significantly impaired lamellipodial formation in both cell lines. Knockdown of AQP1 with siRNA (confirmed by quantitative PCR) reduced the effectiveness of the combined inhibitors, confirming AQP1 as a target of action. Invasiveness measured using transwell filters layered with extracellular matrix in both cell lines was inhibited by AqB011, with a greater potency in HT29 than SW480. A side effect of bacopaside II at high doses was a potentiation of invasiveness, that was reversed by AqB011. Results here are the first to demonstrate that combined block of the AQP1 ion channel and water pores is more potent in impairing motility across diverse classes of colon cancer cells than single agents alone.

Development of a Photoswitchable Lithium-Sensitive Probe to Analyze Nonselective Cation Channel Activity in Migrating Cancer Cells.

This is the first work to use a newly designed Li+-selective photoswitchable probe Sabrina Heng Lithium (SHL) in living colon cancer cells to noninvasively monitor cation channel activity in real time by the appearance of lithium hot spots detected by confocal microscopy. Punctate Li+ hot spots are clustered in the lamellipodial leading edges of HT29 human colon cancer cells and are colocalized with aquaporin-1 (AQP1) channels. AQP1 is a dual water and cyclic-nucleotide-gated cation channel located in lamellipodia and is essential for rapid cell migration in a subset of aggressive cancers. Both the Li+ hot spots and cell migration are blocked in HT29 cells by the AQP1 ion channel antagonist AqB011. In contrast, Li+ hot spots are not evident in a poorly migrating colon cancer cell line, SW620, which lacks comparable membrane expression of AQP1. Knockdown of AQP1 by RNA interference in HT29 cells significantly impairs Li+ hot spot activity. The SHL probe loaded in living cells shows signature chemical properties of ionic selectivity and reversibility. Dynamic properties of the Li+ hot spots, turning on and off, are confirmed by time-lapse imaging. SHL is a powerful tool for evaluating cation channel function in living cells in real time, with particular promise for studies of motile cells or interlinked networks not easily analyzed by electrophysiological methods. The ability to reset SHL by photoswitching allows monitoring of dynamic signals over time. Future applications of the Li+ probe could include high-throughput optical screening for discovering new classes of channels, or finding new pharmacological modulators for nonselective cation channels.

Identification of Loop D Domain Amino Acids in the Human Aquaporin-1 Channel Involved in Activation of the Ionic Conductance and Inhibition by AqB011.

Aquaporins are integral proteins that facilitate the transmembrane transport of water and small solutes. In addition to enabling water flux, mammalian Aquaporin-1 (AQP1) channels activated by cyclic GMP can carry non-selective monovalent cation currents, selectively blocked by arylsulfonamide compounds AqB007 (IC50 170 µM) and AqB011 (IC50 14 µM). In silico models suggested that ligand docking might involve the cytoplasmic loop D (between AQP1 transmembrane domains 4 and 5), but the predicted site of interaction remained to be tested. Work here shows that mutagenesis of two conserved arginine residues in loop D slowed the activation of the AQP1 ion conductance and impaired the sensitivity of the channel to block by AqB011. Substitution of residues in loop D with proline showed effects on ion conductance amplitude that varied with position, suggesting that the structural conformation of loop D is important for AQP1 channel gating. Human AQP1 wild type, AQP1 mutant channels with alanines substituted for two arginines (R159A+R160A), and mutants with proline substituted for single residues threonine (T157P), aspartate (D158P), arginine (R159P, R160P), or glycine (G165P) were expressed in Xenopus laevis oocytes. Conductance responses were analyzed by two-electrode voltage clamp. Optical osmotic swelling assays and confocal microscopy were used to confirm mutant and wild type AQP1-expressing oocytes were expressed in the plasma membrane. After application of membrane-permeable cGMP, R159A+R160A channels had a significantly slower rate of activation as compared with wild type, consistent with impaired gating. AQP1 R159A+R160A channels showed no significant block by AqB011 at 50 µM, in contrast to the wild type channel which was blocked effectively. T157P, D158P, and R160P mutations had impaired activation compared to wild type; R159P showed no significant effect; and G165P appeared to augment the conductance amplitude. These findings provide evidence for the role of the loop D as a gating domain for AQP1 ion channels, and identify the likely site of interaction of AqB011 in the proximal loop D sequence.

Fundamental structural and functional properties of Aquaporin ion channels found across the kingdoms of life.

Aquaporin (AQP) channels in the major intrinsic protein (MIP) family are known to facilitate transmembrane water fluxes in prokaryotes and eukaryotes. Some classes of AQPs also conduct ions, glycerol, urea, CO2 , nitric oxide, and other small solutes. Ion channel activity has been demonstrated for mammalian AQPs 0, 1, 6, Drosophila Big Brain (BIB), soybean nodulin 26, and rockcress AtPIP2;1. More classes are likely to be discovered. Newly identified blockers are providing essential tools for establishing physiological roles of some of the AQP dual water and ion channels. For example, the arylsulfonamide AqB011 which selectively blocks the central ion pore of mammalian AQP1 has been shown to impair migration of HT29 colon cancer cells. Traditional herbal medicines are sources of selective AQP1 inhibitors that also slow cancer cell migration. The finding that plant AtPIP2;1 expressed in root epidermal cells mediates an ion conductance regulated by calcium and protons provided insight into molecular mechanisms of environmental stress responses. Expression of lens MIP (AQP0) is essential for maintaining the structure, integrity and transparency of the lens, and Drosophila BIB contributes to neurogenic signalling pathways to control the developmental fate of fly neuroblast cells; however, the ion channel roles remain to be defined for MIP and BIB. A broader portfolio of pharmacological agents is needed to investigate diverse AQP ion channel functions in situ. Understanding the dual water and ion channel roles of AQPs could inform the development of novel agents for rational interventions in diverse challenges from agriculture to human health.

Divalent Cations Regulate the Ion Conductance Properties of Diverse Classes of Aquaporins.

Aquaporins (AQPs) are known to facilitate water and solute fluxes across barrier membranes. An increasing number of AQPs are being found to serve as ion channels. Ion and water permeability of selected plant and animal AQPs (plant Arabidopsis thaliana AtPIP2;1, AtPIP2;2, AtPIP2;7, human Homo sapiens HsAQP1, rat Rattus norvegicus RnAQP4, RnAQP5, and fly Drosophilamelanogaster DmBIB) were expressed in Xenopus oocytes and examined in chelator-buffered salines to evaluate the effects of divalent cations (Ca2+, Mg2+, Ba2+ and Cd2+) on ionic conductances. AtPIP2;1, AtPIP2;2, HsAQP1 and DmBIB expressing oocytes had ionic conductances, and showed differential sensitivity to block by external Ca2+. The order of potency of inhibition by Ca2+ was AtPIP2;2 > AtPIP2;1 > DmBIB > HsAQP1. Blockage of the AQP cation channels by Ba2+ and Cd2+ caused voltage-sensitive outward rectification. The channels with the highest sensitivity to Ca2+ (AtPIP2;1 and AtPIP2;2) showed a distinctive relief of the Ca2+ block by co-application of excess Ba2+, suggesting that divalent ions act at the same site. Recognizing the regulatory role of divalent cations may enable the discovery of other classes of AQP ion channels, and facilitate the development of tools for modulating AQP ion channels. Modulators of AQPs have potential value for diverse applications including improving salinity tolerance in plants, controlling vector-borne diseases, and intervening in serious clinical conditions involving AQPs, such as cancer metastasis, cardiovascular or renal dysfunction.

Non-selective cation channel activity of aquaporin AtPIP2;1 regulated by Ca2+ and pH.

The aquaporin AtPIP2;1 is an abundant plasma membrane intrinsic protein in Arabidopsis thaliana that is implicated in stomatal closure, and is highly expressed in plasma membranes of root epidermal cells. When expressed in Xenopus laevis oocytes, AtPIP2;1 increased water permeability and induced a non-selective cation conductance mainly associated with Na+ . A mutation in the water pore, G103W, prevented both the ionic conductance and water permeability of PIP2;1. Co-expression of AtPIP2;1 with AtPIP1;2 increased water permeability but abolished the ionic conductance. AtPIP2;2 (93% identical to AtPIP2;1) similarly increased water permeability but not ionic conductance. The ionic conductance was inhibited by the application of extracellular Ca2+ and Cd2+ , with Ca2+ giving a biphasic dose-response with a prominent IC50 of 0.32 mм comparable with a previous report of Ca2+ sensitivity of a non-selective cation channel (NSCC) in Arabidopsis root protoplasts. Low external pH also inhibited ionic conductance (IC50 pH 6.8). Xenopus oocytes and Saccharomyces cerevisiae expressing AtPIP2;1 accumulated more Na+ than controls. Establishing whether AtPIP2;1 has dual ion and water permeability in planta will be important in understanding the roles of this aquaporin and if AtPIP2;1 is a candidate for a previously reported NSCC responsible for Ca2+ and pH sensitive Na+ entry into roots.

Differential Inhibition of Water and Ion Channel Activities of Mammalian Aquaporin-1 by Two Structurally Related Bacopaside Compounds Derived from the Medicinal Plant Bacopa monnieri.

Aquaporin-1 (AQP1) is a major intrinsic protein that facilitates flux of water and other small solutes across cell membranes. In addition to its function as a water channel in maintaining fluid homeostasis, AQP1 also acts as a nonselective cation channel gated by cGMP, a property shown previously to facilitate rapid cell migration in a AQP1-expressing colon cancer cell line. Here we report two new modulators of AQP1 channels, bacopaside I and bacopaside II, isolated from the medicinal plant Bacopa monnieri Screening was conducted in the Xenopus oocyte expression system, using quantitative swelling and two-electrode voltage clamp techniques. Results showed bacopaside I blocked both the water (IC50 117 µM) and ion channel activities of AQP1 but did not alter AQP4 activity, whereas bacopaside II selectively blocked the AQP1 water channel (IC50 18 µM) without impairing the ionic conductance. These results fit with predictions from in silico molecular modeling. Both bacopasides were tested in migration assays using HT29 and SW480 colon cancer cell lines, with high and low levels of AQP1 expression, respectively. Bacopaside I (IC50 48 µM) and bacopaside II (IC50 14 µM) impaired migration of HT29 cells but had minimal effect on SW480 cell migration. Our results are the first to identify differential AQP1 modulators isolated from a medicinal plant. Bacopasides could serve as novel lead compounds for pharmaceutic development of selective aquaporin modulators.

Bumetanide Derivatives AqB007 and AqB011 Selectively Block the Aquaporin-1 Ion Channel Conductance and Slow Cancer Cell Migration.

Aquaporins (AQPs) in the major intrinsic family of proteins mediate fluxes of water and other small solutes across cell membranes. AQP1 is a water channel, and under permissive conditions, a nonselective cation channel gated by cGMP. In addition to mediating fluid transport, AQP1 expression facilitates rapid cell migration in cell types including colon cancers and glioblastoma. Work here defines new pharmacological derivatives of bumetanide that selectively inhibit the ion channel, but not the water channel, activity of AQP1. Human AQP1 was analyzed in the Xenopus laevis oocyte expression system by two-electrode voltage clamp and optical osmotic swelling assays. The aquaporin ligand bumetanide derivative AqB011 was the most potent blocker of the AQP1 ion conductance (IC50 of 14 µM), with no effect on water channel activity (at up to 200 µM). The order of potency for inhibition of the ionic conductance was AqB011 > AqB007 >> AqB006 ≥ AqB001. Migration of human colon cancer (HT29) cells was assessed with a wound-closure assay in the presence of a mitotic inhibitor. AqB011 and AqB007 significantly reduced migration rates of AQP1-positive HT29 cells without affecting viability. The order of potency for AQP1 ion channel block matched the order for inhibition of cell migration, as well as in silico modeling of the predicted order of energetically favored binding. Docking models suggest that AqB011 and AqB007 interact with the intracellular loop D domain, a region involved in AQP channel gating. Inhibition of AQP1 ionic conductance could be a useful adjunct therapeutic approach for reducing metastasis in cancers that upregulate AQP1 expression.