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Challenges and achievements in the therapeutic modulation of aquaporin functionality
Challenges and achievements in the therapeutic modulation of aquaporin functionality Eric Beitz, Andr´e Golldack, Monja Rothert, Julia von B¨ulow PII: DOI: Reference:
S0163-7258(15)00160-6 doi: 10.1016/j.pharmthera.2015.08.002 JPT 6806
To appear in:
Pharmacology and Therapeutics
Please cite this article as: Beitz, E., Golldack, A., Rothert, M. & von B¨ ulow, J., Challenges and achievements in the therapeutic modulation of aquaporin functionality, Pharmacology and Therapeutics (2015), doi: 10.1016/j.pharmthera.2015.08.002
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ACCEPTED MANUSCRIPT Invited Review (edt. Enno Klussmann)
Rev. August 2, 2015
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P & T #22780
Challenges and achievements in the therapeutic modulation of aquaporin functionality
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Eric Beitz, André Golldack, Monja Rothert, Julia von Bülow
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Pharmaceutical and Medicinal Chemistry, University of Kiel, Germany
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Correspondence address:
Eric Beitz Pharmaceutical and Medicinal Chemistry University of Kiel Gutenbergstrasse 76 24118 Kiel, Germany Tel. Fax E-mail:
++49 431 880 1809 ++49 431 880 1352 [email protected]
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ACCEPTED MANUSCRIPT Abstract Aquaporin water and solute channels (AQP) have basic physiological functions throughout
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the human body. AQP-facilitated water permeability across cell membranes is required for rapid reabsorption of water from pre-urine in the kidneys and for sustained near isosmolar
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water fluxes e.g. in the brain, eyes, inner ear, and lungs. Cellular water permeability is further connected to cell motility. AQPs of the aquaglyceroporin subfamily are necessary for lipid degradation in adipocytes and glycerol uptake into the liver, as well as for skin moistening.
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Modulation of AQP function is desirable in several pathophysiological situations, such as
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nephrogenic diabetes insipidus, Sjögren’s syndrome, Menière’s disease, heart failure, or tumors to name a few. Attempts to design or to find effective small molecule AQP inhibitors have yielded only a few hits. Challenges reside in the high copy number of AQP proteins in
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the cell membranes, and spatial restrictions in the protein structure. This review gives an
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overview on selected physiological and pathophysiological conditions in which modulation of
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AQP inhibitors.
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AQP functions appears beneficial and discusses first achievements in the search of drug-like
Keywords: Aquaporin, water, glycerol, volume regulation, inhibition, drug development
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ACCEPTED MANUSCRIPT Table of Contents 1. Introduction
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2. Selected physiological and pathophysiological roles of AQPs 2.1 Kidneys, inner ear
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2.2 Eye, salivary glands 2.3 Central nervous system 2.4 Lungs
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2.5 Tumors
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2.6 Immune system, hematocytes, parasite infections 2.7 Fat metabolism
3. Assay systems for AQP function and inhibition
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3.1 Phenotypic AQP assays
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3.2 Biophysical AQP assays
3.3 High-throughput AQP assays
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4. Towards therapeutic modulation of AQP function
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4.1 AQP protein structures as drug target 4.2 AQP inhibition by transition metals 4.3 AQP inhibition by small organic molecules 4.4 Inhibition of the interaction of AQP4 with autoantibodies 4.5 AQP gene replacement therapy 5. Conclusion
Abbreviations: AQP – aquaporin, NMO – neuromyelitis optica
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ACCEPTED MANUSCRIPT 1. Introduction A survey of the currently used pharmacological agents and classification of their respective
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drug targets puts a figure on the large predominance of compounds that address receptor molecules (44% of all human drug targets; Rask-Andersen et al., 2011). Within the class of
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receptor drug targets almost half represent G-protein coupled receptors and one fifth are ligand-gated ion channel receptors, followed by tyrosine-kinase receptors. The next large target group contains various enzymes (29% of the drug targets) with a major focus on lipid
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mediator-producing oxidoreductases, such as the cyclooxygenases, COX, i.e. the targets of
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nonsteroidal anti-inflammatory drugs. The drug target class of transport and channel proteins ranks third (15% of all drug targets). Here, mainly ion channels of the heart and circular system are modulated in their function to treat arrhythmia and hypertension as well as
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neurotransmitter transporters of the brain for neurological disorders. The new anti-diabetic
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class of gliflozins, that inhibit the renal sodium-dependent glucose transporter, SGLT2, represents one of the very few cases in which high-level transport of a nutrient at millimolar
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concentrations is targeted.
This brief overview shows that the great majority of drugs interfere fairly directly with signal transduction, be it at the receptor, the small-molecule transmitter, or the action potential level. The advantages are obvious: the drugs compete with very low transmitter concentrations, which can be as low as in the femtomolar range, and the target proteins exhibit explicit binding sites, which allow for high-affinity interactions. Both are prerequisites for optimizing drug compounds towards low-dose application and specificity of action.
The situation of transmembrane water transport facilitated by one of the thirteen different human aquaporin channel proteins (AQP0-12) is vastly opposite in both, substrate concentration and affinity aspects. Water represents the most abundant molecule in the human 4
ACCEPTED MANUSCRIPT body (about 60% of the total body mass) and its concentration in the body fluids is 55 molar, i.e. 10,000 times higher than that of the most important energy carrier molecule glucose. With
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respect to substrate affinity, AQP proteins appear inconspicuous to passing water molecules by mimicking the hydrogen bond situation and binding energy of the aqueous bulk. Thus, the
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energy barrier for water permeation through the AQP channel following an osmotic gradient is hardly higher than diffusion in free solution. Structure-wise, AQPs are rigid proteins and exhibit little thermal fluctuations of the amino acid residues in the channel region in order to
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maintain a 20 Å long and very narrow channel pathway of only 2-4 Å in diameter open for
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water (orthodox AQPs; Murata et al., 2000) or small, uncharged solutes, mainly glycerol and chemically resembling compounds (aquaglyceroporins; Fu et al., 2000), posing major space limitations for putative inhibitor molecules. On top of that, AQPs tend to populate the cell
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membranes in large numbers, i.e. the membrane of a single erythrocyte contains about
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200,000 AQP copies (Solomon et al., 1983; Denker et al., 1988).
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Despite the challenges due to the AQP structure and protein abundance, it appears worthwhile
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to search for small molecule modulators due to the many and central roles AQPs play in physiology and pathophysiology. A recent review by Verkman et al. (2014) provides a comprehensive and excellent overview on AQP-related disorders and pharmacological intervention attempts. In this paper, we will focus – after going through physiology and AQPrelated therapeutic possibilities – on options for compound screening, and the protein structural and chemical aspects of AQP modulator design.
2. Selected physiological roles of AQPs and options for modulation Besides water and glycerol, AQPs facilitate permeation, dependent on the isoform, of various other physiological molecules across cell membranes: ammonia, carbon dioxide, urea, hydrogen peroxide, and methylglyoxal (Wu & Beitz, 2007). Potential physiological roles of 5
ACCEPTED MANUSCRIPT AQPs are, thus, in waste metabolite elimination (ammonia, urea, methylglyoxal), cellular gas exchange (carbon dioxide), and oxidative stress relief and/or signal molecule transport
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(hydrogen peroxide). Such AQP functions, if physiologically relevant, have not been attributed to diseases, yet. Hence, in the following, we will summarize data on the more
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classical roles of AQPs that are mainly related to water and glycerol transmembrane transport and in which pharmacological modulation is considered beneficial.
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Lipid bilayers are permeable for water; yet, due to the lipophilic membrane core, osmotic
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diffusion rates are low and considerable activation energy (> 10 kcal mol–1) is required. In the presence of AQP water channels, transmembrane water permeability increases by one to two orders of magnitude and the energetic cost is lowered to that of breaking two to three
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hydrogen bonds (< 5 kcal mol–1; Preston et al., 1992). Two situations call for AQP facilitated
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transmembrane water transport (Fig. 1): a) rapid, high-volume transport (kidneys), and b) sustained water transport at small, near-isosmotic gradients (slow fluid exchange, secretory
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glands, cell motility). Transmembrane transport of glycerol along a chemical gradient is
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relevant in skin moistening and in the Cori cycle, i.e. glycerol release from adipocytes during lipolysis and uptake of glycerol by the liver for gluconeogenesis. We will discuss water transport in the kidneys and the inner ear in one section because in both cases regulation is via vasopressin; thereafter, we address the situation in the eye and surrounding secretory tissues together with water secreting salivary glands.
2.1 Kidneys, inner ear The water transport capacity of the kidneys is unparalleled in the human body. It is driven by a steep osmotic gradient due to the active transport of salt reaching concentrations up to four times higher than in normal tissue. More than 150 liters of blood are filtered by the nephrons per day, equaling 100 ml min–1 or 1.7 ml s–1. This way, hydrophilic, potentially toxic 6
ACCEPTED MANUSCRIPT substances, such as waste metabolites and xenobiotics, are cleared from the body. At the same time, the kidneys regulate the water, salt, and pH homeostasis of the organism. To maintain
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the water-balance, about 99% of the filtered pre-urine water is being reabsorbed. The proximal tubule and the descending thin limb of the Henle loop continuously take up the
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major volume, whereas the remaining, approximately 20% of the kidney filtrate is used to adjust homeostasis via the action of the hormone vasopressin that acts on the water
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permeability of the collecting duct endothelia.
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The discovery of the AQPs eventually provided the molecular basis for the highly water permeable kidney sections. In total, eight AQPs have been localized in different segments of
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the nephron.
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AQP1 is the predominant water channel in the apical and basolateral membranes of the brush border cells of the proximal tubules, in the loop of Henle, as well as in the endothelium of the
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descending vasa recta (Nielsen et al., 1993b). Accordingly, AQP1-null mice suffer from a
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major reduction in water reabsorption in the proximal tubules (Ma et al., 1998; Schnermann et al., 1998; Chou et al., 1999; Pallone et al., 2000). HgCl2 and organo-mercurials are known AQP1 inhibitors that bind to a cysteine residue (Cys189 in human AQP1) close to the extracellular pore entry. Such mercurials were used as diuretics until the 1950s (Nielsen et al., 1999) and AQP1 is one putative site of action. Due to the highly unspecific nature of cysteine-modification by mercurials it is very likely, though, that other protein components of the kidney water reabsorption system, e.g. ion channels, were hit as well. Likewise, other AQPs carrying a cysteine in the pore region are inhibited by mercurials. The co-incidence that AQP1 is additionally present in red blood cell membranes and exposes the Colton blood group epitope, led to the identification of women lacking AQP1 altogether. Their urine concentration defect became evident only when water drinking was restricted whereas with 7
ACCEPTED MANUSCRIPT unrestricted fluid uptake no polyuria was noticeable suggesting compensation by other AQPs
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or unknown mechanisms present in the human kidney (King et al., 2001).
AQP2 is a second major kidney water channel present in the principal cells of the collecting
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duct (Fushimi et al., 1994) where it acts as an indirect drug target of the new class of vaptans, i.e. vasopressin type 2 receptor antagonists (Villabona, 2010). Under basal conditions, AQP2 is stored in perinuclear vesicles, which translocate, upon vasopressin-elicited cAMP signaling
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and phosphorylation of AQP2 in the C-terminal region by protein kinase A, to the apical
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plasma membrane (Nielsen et al., 1993a; Brown, 2003; Sasaki, 2012). Incorporation of AQP2 into the plasma membrane increases water permeability and, thus, reabsorption of pre-urine. Functional defects in the vasopressin type 2 receptor or in AQP2 result in a daily production
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of up to 20 liters of a dilute urine, i.e. nephrogenic diabetes insipidus (Deen et al., 1994;
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Brown, 2003; Kamsteeg et al., 2003; Nguyen et al., 2003). Mouse models lacking kidney AQP2 (Rojek et al., 2007) or expressing an intracellularly misrouted AQP2 variant
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(Thr126Met; Yang et al., 2001) confirmed the role of AQP2 in vasopressin-dependent urine
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volume regulation. The phenotype of the latter could be partially corrected by an inhibitor of heat-shock protein 90 (Yang et al., 2009). Nephropathies induced by lithium, hypokalemia, or by cisplatin treatment can manifest as forms of nephrogenic diabetes insipidus and appear to be associated with decreased expression of AQP2 (Nielsen et al., 2002). Vaptans as inhibitors of the vasopressin/AQP2-dependent water reabsorption act as aquaretics (increasing the secreted water volume) rather than as diuretics (increasing salt and water secretion; Villabona, 2010). Vaptans are used to treat clinical conditions associated with water retention, such as in congestive heart failure, or liver cirrhosis (Schrier et al., 1998; Nielsen et al., 2002), as well as euvolemic and hypervolemic hyponatremia (Izumi et al., 2014). Specific direct inhibitors of kidney AQP1 or AQP2 should be applicable for the same indications.
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ACCEPTED MANUSCRIPT Vaptans have further been successfully used to treat an animal model of surgically induced inner ear hydrops (Takeda et al., 2003). The molecular setup of the inner ear endolymph
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volume regulation system appears to be highly similar to that of the kidney and includes the vasopressin type 2 receptor and AQP2 (Kumagami et al., 1998; Beitz et al., 1999).
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Endolymph hydrops, i.e. an overpressure in the endolymph compartment harboring the sensory cells for hearing and balance, is responsible for the symptoms of Menière’s disease,
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hearing loss and severe vertigo attacks.
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Of the remaining six kidney AQPs, AQP3 and AQP4 co-localize with AQP2 in the collecting duct but are constitutively present in the basolateral membranes of the principal cells where they form the exit pathways for reabsorbed water into the tissue (Nielsen et al., 2002). AQP3
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knockout mice exhibit a polyuric and polydipsic phenotype comparable to that of AQP1 (Ma
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et al., 2000). AQP4 accounts for 75 % of basolateral water transport in mice, yet deletion of the AQP4 gene reduced the ability to concentrate urine only moderately (Ma et al., 1997).
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Recently, AQP5 was localized in type B intercalated cells of the collecting duct (Porcino et al.
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2011). Since the AQP5 is present in the apical membrane and the basolateral membrane lacks AQPs it was speculated that the cells may act in osmosensing. AQP6 is expressed in acidsecreting intercalated cells of collecting ducts and co-localizes intracellularly with the vacuolar H+-ATPase (Yasui et al., 1999; Ikeda et al., 2002). AQP6 is permeable to anions, such as nitrite and chloride, and is gated by pH suggesting a role in vesicle acidification; however, the phenotype of an AQP6 knock-out mouse is still elusive. AQP7 and AQP8 are expressed in the proximal tubule of the nephron (Nielsen et al., 2002), but a renal function has not been attributed to these isoforms, yet. The knockout of AQP11 in mice, however, leads to a severe and lethal phenotype of massive vacuolization and cyst formation in the proximal tubules (Morishita et al., 2005); the animals die within two months after birth. AQP11 is localized intracellularly in the endoplasmic reticulum from which the vacuolization process is 9
ACCEPTED MANUSCRIPT initiated when AQP11 is absent (Okada et al., 2008). The resemblance of cyst formation in the knockout mice and in polycystic kidney disease models may help elucidating the
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underlying molecular mechanisms leading to the disease.
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2.2 Eye, salivary glands
The sensory cells of the eye’s retina are situated in an enclosed fluid-filled chamber, which requires, similar to the inner ear, careful volume regulation by AQPs to adjust the intraocular
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pressure (Frigeri et al., 1995). AQP1 and AQP4 are expressed in the iris and the ciliar
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epithelium and are thought to carry the major portion of chamber water production (Zhang et al., 2002). Increased intraocular pressure, glaucoma, poses stress to the cells of the retina and the optic nerve (Huber et al., 2012). There are pharmacological agents for the treatment of
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both, wide-angle and narrow-angle glaucoma, and inhibitors of AQP1 and AQP4 may become
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alternatives to adjust water regulation in the eye.
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AQP water channels are further central for the maintenance of cornea and lens transparency
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(Verkman, 2003), e.g. corneal thickness is decreased in AQP1-null mice (Huber et al., 2012). This example and the fact that AQP1 is generally the most widely distributed AQP throughout the human body should be considered with respect to putative side effects when thinking about systemic inhibition of AQP1. AQP0 of the eye lens was found to have low waterpermeability, its main function appears to be structural and to reside in the parallel fixation of cells by cell-cell contacts (Mulders et al., 1995). Mutations in AQP0 have been associated with human congenital cataract (Berry et al., 2000; Geyer et al., 2006). Correction of such structural effects by pharmacologically means seems difficult; yet, the accessibility of the cornea and lens may permit gene therapeutic approaches to replace defect AQP0 in the future.
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ACCEPTED MANUSCRIPT AQP3, found in the conjunctiva, and AQP5, in lacrimal glands, are required for the secretion of eye surface liquids (King et al., 2004). Respective agonists may be beneficial in treating
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dry eye syndrome. Patients with Sjøgren´s syndrome suffering from dry eyes and mouth have been identified to carry mutations in AQP5 that lead to intracellular misrouting of the water
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channel in the lacrimal and salivary glands (Steinfeld et al., 2001; Tsubota et al., 2001). A causative therapy would have to re-establish proper AQP5 levels and localization to the apical
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membranes in the affected cells.
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2.3 Central nervous system
Changes in the volume of the cerebrospinal fluid directly pose stress on the brain cells whereas changes in the ionic composition of the fluid can alter neuronal signaling. Seven
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AQPs were amplified from cDNA of mammalian brain cells; however, on the protein level,
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only three AQPs could be confirmed of which AQP1 and AQP4 predominate (Badaut et al., 2007), whereas AQP9 protein levels are low and knockout mice do not exhibit a cerebral
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phenotype. AQP1 is found in the choroid plexus epithelium (Nielsen et al., 1993c) where it
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facilitates secretion of the cerebrospinal fluid and regulation of the intracranial pressure (Oshio et al., 2005); inhibition of AQP1 may be an option for the treatment of hydrocephalis (Tait et al., 2008). AQP1 is also present in neuronal cells, such as nociceptive C-fibers (Oshio et al., 2004; Oshio et al., 2006; Shields et al., 2007) and dorsal root ganglion neurons (Zhang & Verkman, 2010). An involvement of AQP1 water permeability in pain sensing is discussed and AQP1 inhibitors may even turn out as a novel principle in analgesic therapy.
AQP4 is the most abundant AQP in the human brain and is predominantly found in the basolateral membrane of ependymal cells, and in astrocytes interfacing the cerebrospinal fluid compartment and the blood (Nielsen et al., 1997; Rash et al., 1998). AQP4 knockout mice were better protected from brain edema induced by water intoxication or cerebral ischemic 11
ACCEPTED MANUSCRIPT injury than wild-type animals and exhibited less neurological damage and lower rates of mortality suggesting a putative role of AQP4 inhibitors in the acute treatment of such edema
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(Manley et al., 2000; Papadopoulos & Verkman, 2005; Haj-Yasein et al., 2011; Katada et al., 2014). Oppositely, AQP4 appears to be responsible for the volume clearance in vasogenic
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brain edema that is caused by increased permeability of the blood brain barrier (Papadopoulos et al., 2004). AQP4 null mice suffered from increased intracranial pressure giving a worsened clinical outcome compared to wild-type mice, when vasogenic edema was induced by
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continuous intracerebral fluid infusion, freeze-injury, an intraparenchymal bacterial abscess,
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or brain tumors (Bloch et al., 2005; Papadopoulos et al., 2004; Papadopoulos & Verkman, 2005). Accordingly, inhibition of AQP4 would require secure diagnosis of the underlying cause of the edema. Besides water regulation, neuronal activity of AQP4 null mice was
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affected as seen by a higher seizure threshold and prolonged seizure duration, which may be
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beneficial in epilepsy therapy (Binder et al., 2006); at the same time, mice lacking AQP4 are deaf, again pointing to severe potential side effects of AQP4 inhibitors (Li & Verkman,
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2001).
AQP4 is also associated with neuromyelitis optica (NMO), an inflammatory autoimmune disease of the optic nerve and the spinal cord. 60-90% of the patients generate autoantibodies that are directed against AQP4 and cause complement and immune cell-mediated damage to astrocytes (Jarius & Wildemann, 2010; Lennon et al., 2005). Current developments aim at the inhibition of the interaction of AQP4 and the autoantibodies and are discussed below in section 4.4.
2.4 Lungs Water transport across epithelial and endothelial barriers in bronchopulmonary tissues occurs during airway hydration, alveolar fluid transport, and submucosal gland secretion (Borok & 12
ACCEPTED MANUSCRIPT Verkman, 2002). AQP1, AQP3, AQP4 and AQP5 are expressed in the lung and are candidates for facilitating respective water transport (Verkman, 2007). Human AQP1-null
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individuals showed thickening of the airway walls, i.e. signs of peribronchiolar edema, after ingestion of large quantities of fluid (King et al., 2002). In mouse studies, however, deletion
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of genes encoding AQP1, AQP3, or AQP4 did not produce a lung phenotype. Solely, AQP5null mice showed impaired fluid secretion by airway submucosal glands and subsequent changes in the composition, viscosity, and volume of the airway surface liquid (Song &
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Verkman, 2001). Therapeutic modulation of AQP5 on the transcriptional or functional level,
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thus, could be meaningful in cystic fibrosis (Verkman, 2007).
2.5 Tumors
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Different AQPs have been found in various tumors and related cell types (Saadoun et al.,
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2002b; Saadoun et al., 2002a). Their role apparently does not solely reside in keeping up water balance as laid out above, but in bringing about cellular functions regarding migration,
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proliferation, and adhesion (Verkman et al., 2014), which are prerequisites for tumor growth, angiogenesis, metastasis, and tissue infiltration. The field of AQP-related tumor biology is
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rapidly expanding; hence, we will highlight some examples and refer to specific recent reviews for further reading (Papadopoulos & Saadoun, 2014; Ribatti et al., 2014; Verkman et al., 2014).
Research in the field was ignited by the notion that in AQP1-null mice subcutaneously or intracranially implanted tumors grew markedly slower, as did breast tumors and lung metastases (Saadoun et al., 2005; Esteva-Font et al., 2014). The growth defect was due to increased necrosis and reduced vascularisation. Typically, AQP1 is found in respective microvessels (Endo et al., 1999; Vacca et al., 2001). Primary cultures of vessel endothelial cells from AQP1-null mice showed unaltered adhesion and proliferation; however, cell 13
ACCEPTED MANUSCRIPT migration was largely impaired and in vitro vessel formation was abnormal (Saadoun et al., 2005). Inversely, overexpression of AQP1 in transfected tumor cells increased cell migration
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2-3 fold (Hu & Verkman, 2006). The molecular role of AQP water permeability in cell migration is thought to reside in facilitating the formation of membrane protrusions at the
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leading edge (Papadopoulos et al., 2008). As a driving force, a local increase in the osmolarity by actin depolymerisation and ion transport initiates water influx (Condeelis, 1993; Lauffenburger & Horwitz, 1996; Schwab, 2001; Verkman, 2005). Inhibitors of AQP water
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permeability, thus, may be useful in anti-tumor therapy by interfering with tumor
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angiogenesis and tumor cell spreading. An apparently promising attempt was made in a nude mouse colon cancer model, which was treated with acetazolamide that was found to reduce
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AQP1 expression (Bin & Shi-Peng, 2011).
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High AQP5 expression levels have been associated with a worsened prognosis in non-small cell lung cancer (Chae et al., 2008). In vitro assays indicated that elevated AQP5 expression
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enhanced cell proliferation, migration, and invasion (Zhang et al., 2010). AQP5 was also
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found to promote proliferation and migration of human gastric carcinoma cells (Huang et al., 2013). Hence, AQP5 has been suggested as a prognostic cancer marker (Jung et al., 2011). AQP5 inhibitors might also be useful as anticancer therapeutics.
AQP3 is the major aquaglyceroporin in barrier tissues, such as the skin, (Matsuzaki et al., 1999) and lack of AQP3 affects hydration, elasticity, wound healing, and barrier function (Hara & Verkman, 2003). Elevated AQP3 expression levels are found in squamous cell skin carcinomas, whereas AQP3-null mice exhibit slower tumor growth and spreading (HaraChikuma & Verkman, 2008a, 2008b). The lack of glycerol permeability via AQP3 leads to a reduced cellular glycerol content and ATP biosynthesis, resulting in slow cell proliferation. Accordingly, therapeutic AQP3 inhibitors might interfere with skin tumor formation and 14
ACCEPTED MANUSCRIPT growth. Currently, cosmetics are marketed that contain inducers of AQP3 expression in keratinocytes in order to improve skin hydration. It is unclear at the moment whether and, if
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so, by what extent an increase in the carcinogenic risk is given when such cosmetics are used (Verkman, 2008). AQP3 was further shown to facilitate growth of human esophageal and oral
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squamous cell carcinoma (Kusayama et al., 2011). In this context, downregulation of AQP3 by siRNA increased the therapeutic effect of cisplatin and led to higher sensitivity of prostate
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cancer cells to experimental cryotherapy (Ismail et al., 2009).
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2.6 Immune system, hematocytes, parasite infections
Motility and rapid changes in cell volume are hallmarks of activated cells of the immune system. Dendritic cells are highly motile during antigen presentation and express AQP3 and
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AQP7 (Moon et al., 2004). AQP3 null mice exhibit developmental shifts and reduced motility
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in the dendritic cell subpopulations (Song et al., 2011). B and T lymphocytes initiate expression of AQP1, AQP3, and AQP5 upon activation, whereas AQP3 is already present in
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immature dendritic cells (Moon et al., 2004). In macrophages that have been activated by
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inflammation signals, deletion or inhibition of AQP1 results in reduced secretion of interleukin 1 (Rabolli et al., 2014). Similar to the work on AQPs in tumor biology, investigation of the role of AQPs in the immune response is just taking up speed. Since AQPs seem to fulfill roles mainly in activated and migrating immune cells it seems fair to propose that research on AQP inhibitors may lead to new approaches in anti-inflammatory therapy.
AQP9 function is linked to the treatment of a certain form of leukemia, i.e. acute promyelocytic leukemia; here, it is not water transport that is relevant but provision of an entry pathway for the drug arsenic trioxide, As2O3. When dissolved in water the compound forms As(OH)3 resembling in shape the glycerol molecule, which is a permeant of AQP9. Once inside the cytosol, arsenic drives the malignant promyelocytes into apoptosis (Dong, 15
ACCEPTED MANUSCRIPT 2002). The treatment possibilities by arsenic of other types of cancer including solid tumors are under investigation. In this regard, a lung adenocarcinoma cell line was identified that was
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resistant to arsenic trioxide due to downregulation of AQP9 expression (Lee et al., 2006).
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Pentavalent organic metalloids containing arsenic or antimony also are still the first line treatment of infectious diseases caused by Leishmania parasites, i.e. leishmaniasis (Mukhopadhyay et al., 2011). The compounds will be reduced to trivalent hydroxides by the
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human host cell and taken up by the parasite via an aquaglyceroporin, LmAQP1 in the case of
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Leishmania major (Gourbal et al., 2004; Mukhopadhyay et al., 2011). Resistant Leishmania strains have been isolated and are characterized by reduced AQP expression levels. Similarly, an aquaglyceroporin of Trypanosoma brucei parasites, TbAQP2 (Uzcategui et al., 2004),
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causing trypanosomiasis is required for drug uptake of pentamidine and melarsoprol (Alsford
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et al., 2012; Baker et al., 2012). It remains to be clarified how exactly the large and in the case
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transported.
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of pentamidine positively charged compounds make use of TbAQP2 in order to be
Aquaglyceroporins generally appear to predominate over water-specific AQPs in humanpathogenic parasites causing Chagas´ disease (Trypanosoma cruzi; Montalvetti et al., 2004), toxoplasmosis (Toxoplasma gondii; Pavlovic-Djuranovic et al., 2003), and malaria (Plasmodium spp.; Hansen et al., 2002). Their cellular functions are thought to reside in the compensation of osmotic stress, e.g. during kidney passages and transmission, in the release of waste metabolites, such as ammonia (Zeuthen et al., 2006) and methylglyoxal (PavlovicDjuranovic et al., 2006), and in the uptake of glycerol as a metabolic precurser for glycerolipid biosynthesis (Beitz et al., 2004; Song et al., 2012). Accordingly, they hold a good potential as novel drug targets.
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ACCEPTED MANUSCRIPT 2.7 Fat metabolism The human aquaglyceroporins AQP7 and AQP9 are expressed in adipose tissue and the liver,
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respectively (Hara-Chikuma et al., 2005; Kuriyama et al., 2002). AQP7 facilitates glycerol efflux from adipocytes during lipolysis (Maeda et al., 2004). As a consequence, AQP7 null
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mice become obese and insulin resistant due to functioning lipid accumulation but dysfunctional lipid degradation and release (Hibuse et al., 2005). In humans, mutations of AQP7 are associated with an inability to elevate plasma glycerol levels during exercise
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(Kondo et al., 2002). Increasing the glycerol permeability of adipocytes by acting on AQP7 or
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by other means might provide a novel access to fat reduction in obesity (Wang et al., 2006).
AQP9 can be considered as the liver counterpart of AQP7 in that it is responsible for the
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uptake of glycerol from the plasma, which is used for gluconeogenesis (Jelen et al., 2011;
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Jelen et al., 2012). In AQP9 null mice, plasma levels of glycerol and glycerolipids are increased and liver glucose biosynthesis is independent from plasma glycerol levels (Rojek et
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al., 2007; Jelen et al., 2011). Inhibition of AQP-facilitated glycerol uptake by the liver may be
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beneficial in the prevention of steatosis and consequences, such as steatohepatitis and cirrhosis (Calamita et al., 2012; Rodríguez et al., 2014).
The selection of examples described above show that, from a (patho-)physiological perspective, AQPs are promising drug targets. In order to test small molecule inhibitors permeability assay systems are required that are reliable, easy to handle, and open to upscaling processes in terms of increased throughput for screening larger compound libraries.
3. Assay systems for AQP function and inhibition
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ACCEPTED MANUSCRIPT Determination of water or solute permeability is demanding and requires formation of two compartments separated by a membrane carrying the channel or transport protein of interest
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(Fig. 2). Compartmentation can be achieved by employing living cells in assay media or by using artificial systems, such as proteoliposome suspensions or black lipid membranes
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separating two buffer reservoirs. The establishment of an osmotic or chemical gradient initiates AQP-facilitated water or solute flux, respectively, across the membrane. Phenotypic effects, e.g. cell-growth, or biophysical measures, such as light scattering, fluorescence,
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radiotracer decay, or surface plasmon resonance, serve as a readout. AQP inhibitors can be
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found by adding test compounds prior to the assay. The challenge lies in the construction of a
3.1 Phenotypic AQP assays
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robust setup that is ready for automation and high-throughput screenings.
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To expose an AQP-dependent growth phenotype, yeast cells can be challenged by osmotic stress, forced to take up toxic compounds, or provided access to nutrients (Fig. 2A). Yeast
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strains are rendered osmo-sensitive and cease to grow under hypertonic conditions when an
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aquaglyceroporin is expressed and endogenous glycerol acting as osmotic pressure regulating compatible solute cannot accumulate (Pettersson et al., 2006). Yeast growth will also be affected if the AQP facilitates uptake of toxic arsenite (Liu et al., 2002; Wu et al., 2010), methylamine (Beitz et al., 2006; Zeuthen et al., 2006), or hydrogen peroxide (Bienert et al., 2007; Almasalmeh et al., 2014). A positive growth selection of yeast is possible by heterologous expression of an ammonia-facilitating AQP in a strain that lacks endogenous ammonium transporters (Holm et al., 2005). Phenotypic growth assays can be carried out in the presence or absence of putative AQP inhibitors either on solid agar media with photographic imaging of the cell density (Wu et al., 2008) or in liquid cultures by documenting the turbidity over time (Almasalmeh et al., 2014). The latter yields more quantitative data when integrating the areas under the curves from which IC50 values can be 18
ACCEPTED MANUSCRIPT estimated. A limiting factor of phenotypic yeast growth assays is the incubation time of several days during which added compounds are likely to be lost due to chemical instability
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or metabolism by yeast enzymes. Further, each compound has to be analyzed with respect to
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cytotoxicity in order to recognize false positives.
In this sense, biophysical assays appear more suitable for compound screening because measurements are carried out on a much shorter, seconds to minutes timescale. However, they
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require sophisticated instrumentation and sample handling, which hamper automation and
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throughput.
3.2 Biophysical AQP assays
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The first cell-based biophysical assays for water permeability of specific AQPs were carried
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out using Xenopus laevis oocytes, which were injected with cRNAs encoding the AQP of interest (Preston et al., 1992). Within four days maximal protein levels are obtained and the
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oocytes are challenged by a hypotonic shock or by isotonic replacement of salt from the
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buffer by a solute to test for instance for glycerol permeability. Oocyte swelling due to water influx will be video-monitored and the rate is calculated from the increase of the area covered by the oocyte (Fig. 2B). The large size of the spherical oocytes of approximately 1.2 mm in diameter yields relatively low swelling rates requiring monitoring times of around one minute (Beitz et al., 2009). Parallelization is thinkable by placing the oocytes in 96-well plates and imaging the whole set-up. However, the preparation procedure of the Xenopus oocytes, i.e. operation on a female frog, selection of mature stages, proteolysis of the surrounding vitelline layer, and individual cRNA injection limits higher throughput screening.
The transition to easier to obtain, yet smaller cells, such as cultured epithelial cells, with a width in the range of 10-20 µm and a flat shape, or to erythrocytes, yeast cells, or 19
ACCEPTED MANUSCRIPT proteoliposomes of a few hundred nanometers in diameter calls for more rapid (seconds and
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subseconds range) and precise determination of volume changes.
Suspensions of cells or proteoliposomes can be analyzed using a stopped-flow apparatus by
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recording the particle size-dependent intensity of scattered light (Fig. 2B). The cells or proteoliposomes are rapidly mixed with an osmolyte or solute containing buffer and data acquisition is run for several seconds. Volume increase results in a decrease of the light
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scattering intensity whereas shrinkage leads to elevated signals. Human erythrocytes are well
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suited for this assay because they form non-adherent cell suspensions and contain high levels of water-specific AQP1 and the aquaglyceroporin AQP3 in their native membrane environment facilitating direct evaluation of respective modulators (Martins et al., 2012). The
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number of AQP1 monomers in human erythrocytes was estimated via quantitative
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immunoblotting to 200,000 monomers per erythrocyte (Denker et al., 1988). This lies in the same range as the number of water channels estimated by biophysical analysis of 270,000
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(Solomon et al., 1983). Heterologously expressed AQPs in baker’s yeast, Saccharomyces
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cerevisiae, are a reasonable alternative. Enzymatic zymolyase digestion of the rigid yeast cell wall prior to the assay provides the required elasticity of the obtained protoplasts for the assay (Bertl et al., 1998). Generally, the stopped-flow light scattering technique provides the necessary accuracy and reproducibility to determine IC50 or Ki values. However, the devices typically hold a single measuring chamber and are, thus, better suited for careful evaluation of candidate inhibitors than for running large screening programs (Levin et al., 2007).
Most native human cells are adherent and examination of volume changes is possible by microscopy (Fig. 2B). Since the trace length of a beam of light through a cell layer is a onedimensional indicator of the cell volume, interferometric microscopy can be used to follow volume changes of AQP expressing cells (Farinas & Verkman, 1996). Alternatively, 20
ACCEPTED MANUSCRIPT recordings of volume-dependent fluorescence intensity of entrapped fluorophores can be used to monitor cell swelling and shrinking (Farinas et al., 1995; Soveral et al., 2007; Mola et al.,
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2009; Madeira et al., 2010). In this sense, the chloride-sensitive yellow fluorescent protein, YFP-H148Q/V163S indicates cell swelling via dilution of cytoplasmic chloride
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concentrations and decreasing fluorescence intensity (Galietta et al., 2001; Baumgart et al., 2012; Esteva-Font et al., 2013).
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If epithelial cells form a tight layer, transcellular water flux can be quantified using an Ussing
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chamber (Fig. 2B). Loss and gain of volume on the apical and the basolateral sides of the cell membrane as a result of a transepithelial osmotic gradient can be measured as changes in the electrical conductivity and converted into a fluid transport rate (µl cm–2 h–1) (Edelman &
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Miller, 1991). A more artificial, yet, pure lipid-protein setup, i.e. black lipid membranes,
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involves a single lipid bilayer facing a buffer chamber on either side (Fig. 2B). Ag/AgCl electrodes register electrolyte dilutions close to the bilayer due to water flux via reconstituted
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AQPs as changes in the electrochemical potentials (Bárány-Wallje et al., 2005). The solute
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permeability of the aquaglyceroporin of malaria parasites has been analyzed using surfacetethered proteoliposomes. The process of solute loading of the proteoliposomes led to changes is the refractive index and was measured by surface plasmon resonance (Brändén et al., 2010).
3.3 High-throughput AQP assays In order to identify hits from a high-throughput inhibitor screening a definitive yes/no statement from single time point measurements is desired. One successful approach for identifying inhibitors of the urea transporter type B, UT-B, employed native, AQP1containing erythrocytes in an assay with cell lysis as a readout (Levin et al., 2007). The erythrocytes were preloaded in hypertonic acetamide solution and subsequently exposed to an 21
ACCEPTED MANUSCRIPT isotonic buffer without the compound resulting in acetamide efflux due to the chemical outward gradient. In the presence of a UT-B inhibitor, the entrapped acetamide maintained
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the increased intracellular osmolarity and led to water influx via AQP1 and eventually cell lysis. This way, a library of about 50,000 small molecules was screened. Inhibitors of AQP1
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should be identifiable in a similar way by monitoring the resistance towards erythrocyte lysis in hypotonic buffers (Table 1).
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A variation of the erythrocyte lysis assay was recently applied to yeast cells. Here the AQP-
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dependent freeze tolerance of the cells was used as readout (Ahmadpour et al., 2014; To et al., 2015). Yeast cells expressing a water-permeable AQP recover better from shock freezing and thawing, which can be monitored within hours by a spectroscopic viability assay. AQP
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inhibitors would be identified by reduced viability. The yeast system reproduced the findings
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of an AQP1 inhibitor screen based on microscopy of human cells (Mola et al., 2009) and
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and costs.
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appears quite attractive with respect to free selection of the AQP to be expressed, robustness,
4. Towards therapeutic modulation of AQP function The ability of small molecules to cause a pharmacological effect is based on the interaction with their target proteins. The physicochemical properties of both, small molecule and target, determine whether the interaction is of sufficient specificity and affinity in order to generate the desired efficacy and to reduce the risk of unwanted side effects. Interaction sites of small molecules are based on the type and spatial orientation of their functional groups and usually form hydrogen bonds, and ionic, hydrophobic, and cation- interactions; covalent suicideinhibitors are rare. The affinity rises with the number of interactions, the rigidity of the small molecule, and with the degree of the shielding from the aqueous solvent because water molecules will compete for the interaction sites within a protein. Hence, a deep binding 22
ACCEPTED MANUSCRIPT pocket that is optimally filled by a drug substance produces the best affinity. It further turned out that it is favorable if the compound provides no more than 5 hydrogen bond donor and 10
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hydrogen bond acceptor sites and if the molecular mass is less than 500 Da (rule of five;
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Lipinski et al., 2001).
In order to become useful as a drug substance, the small molecule has to comply with certain pharmacokinetic requirements. First of all, it is necessary that the compound reaches the
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target. While only a few compounds can enter circulation via transport proteins, the majority
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of compounds will have to exhibit sufficient lipophilicity to enable transmembrane diffusion. A measure for the lipophilicity of drug substances is the partition-coefficient, P, representing the equilibrium concentration ratio of the compound in 1-octanol and water. The diffusion of
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small molecules through the tissue is proportional to the logarithm of the partition-coefficient
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described by logP. Drug substances with a good oral bioavailability and good distribution
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behavior usually exhibit a logP smaller or close to 5 (Lipinski et al., 2001).
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4.1 AQP protein structures as targets for small molecules Since the AQP protein family is ancient and shares a highly conserved fold especially in the central parts of the conducting channel, finding a specific small molecule inhibitor could be demanding. AQPs form homotetramers with four individual water/solute conducting channels in the protomers. Two filter regions are located within the channel interior, i.e. the aromatic/arginine (ar/R) selectivity filter towards the extracellular pore entry and the central Asn-Pro-Ala (NPA) region (Fig. 3, top). The ar/R region represents the narrowest constriction and selects permeants by size (Beitz et al., 2006). Together with the NPA region inorganic cations and protons are perfectly prevented from leaking through the AQP (Wu et al., 2009; Wree et al., 2011). The inflexible and tight, funnel-shaped inner structure leaves only little space for potential inhibitors (Fig. 3A). At the same time, it is necessary that an inhibitor 23
ACCEPTED MANUSCRIPT binds with high affinity to displace the excessively abundant water molecules. Affinity is increased if interactions with the protein are shielded against water by lipophilic moieties of
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the inhibitor. However, in this case it is likely that the compound would be oversized to fit the AQP channel. Generally, aquaglyceroporins appear to have deeper and wider vestibules than
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water-specific AQPs (Fig. 3, bottom).
An accompanying table to the text below lists candidate compounds, concentration ranges,
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test systems, and outcome with respect to AQP inhibition (Table 1).
4.2 AQP inhibition by transition metals
Long before the discovery of the AQPs, it was found that mercuric chloride and
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organomercurials inhibit water and solute permeability of native erythrocytes (Fig. 4; Macey
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& Farmer, 1970). In fact, this notion hinted at the proteinaceous nature of the elusive water channel and further boosted the search for the AQPs. Later, direct inhibition of the isolated
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erythrocyte water channel by mercuric chloride was shown in Xenopus oocytes expressing
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human AQP1 (Preston et al., 1992) and the mercurial-sensitive site was located to Cys189 at the extracellular pore constriction (Preston et al., 1992; Preston et al., 1993; Zhang et al., 1993; Ozu et al., 2011). AQP2 is equally sensitive to mercurials and also carries a cysteine at the respective position (Fushimi et al., 1993), whereas AQP4 with an alanine at the critical site was identified as a first mercurial-insensitive water channel (Hasegawa et al., 1994). AQP4, when reconstituted in proteoliposomes, however, exhibited mercurial-sensitivity suggesting participation of a cysteine at the intracellular surface (Yukutake et al., 2008). Other metal-based compounds containing silver, gold, copper, nickel, lead, or tin were found to inhibit aquaporins as well (Niemietz & Tyerman, 2002; Zelenina et al., 2003; Zelenina et al., 2004; Yang et al., 2006; Mola et al., 2009). The coordination gold(III) complexes auphen (Fig. 4), audien, aubipy, and auterpy selectively inhibit glycerol and water transport in red 24
ACCEPTED MANUSCRIPT blood cells via the aquaglyceroporin AQP3 (without affecting AQP1) at low micromolar concentrations (Martins et al., 2012; Martins et al., 2013). Docking models as well as
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mutagenesis identified Cys40 located towards the extracellular face of AQP3 to coordinate the gold complexes, whereas Cys189 in AQP1 (Fig. 3A) appeared inaccessible for these
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larger compounds (Martins et al., 2012; Martins et al., 2013; Serna et al., 2014). The same gold(III) complexes where found to inhibit also AQP7 in adipocytes (Madeira et al., 2014), however in this case other binding modes for gold(III) should be in place since this isoform
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lacks Cys40. Thus, binding to Cys residues is not always the primary mechanism of inhibition
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for metal-based compounds, but others may be in place and further investigation is necessary.
Metallo-compounds are the most effective AQP inhibitors available. This is certainly due to
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the high affinity of a metal-thiolate complex which is partially covalent yielding binding
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energies well above 100 kcal mol–1, i.e. approximately one third that of a fully covalent bond (Solomon et al., 2006). The affinity problem in AQP inhibition seems, thus, solved. Using
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such potent binders, however, one has to make sure that high selectivity is given in order to
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prevent quasi-permanent modification of uninvolved proteins and the risk of side effects. Hence, effects of simple inorganic mercuric chloride and salts of the other mentioned metals are a topic in toxicology but certainly not in pharmacology. A larger scaffold in the organic part of the metal-complex, as it is given in the auphen-like compounds, may provide options for molecule modifications that lead to the required selectivity (Martins et al., 2012). There are established examples of heavy metal-based compounds, such as auranofin in the treatment of rheumatoid arthritis, or the anticancer drug cisplatin showing that, in principle, metallodrugs are of therapeutic value (Mjos & Orvig, 2014).
4.3 AQP inhibition by small organic molecules
25
ACCEPTED MANUSCRIPT The outcome of studies on non metal-based small molecule inhibitors targeting AQPs is mixed and apparently depends on the assay system. One of the first compounds for which
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AQP inhibition was published is the quaternary nitrogen compound tetraethylammonium (TEA; Brooks et al., 2000; Detmers et al., 2006). Experiments with AQP-expressing Xenopus
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oocytes yielded significant, dose dependent and reversible inhibition of water permeability of AQP1, AQP2, and AQP4 (Brooks et al., 2000; Detmers et al., 2006). Additionally, the tetraethylammonium analogue tetrapropylammonium (TPrA) was effective on AQP1
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suggesting that quaternary nitrogen compounds represent a class of AQP blockers. Mutational
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studies identified Tyr186 of the extracellular loop E of AQP1 to be required for tetraethylammonium-inhibition (Brooks et al., 2000; Detmers et al., 2006; Müller et al., 2008). At 4 µM, tetraethylammonium inhibited AQP1 water permeability of Xenopus oocytes
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by 46%. Inhibition was also seen in AQP1-expressing Madin-Darby canine kidney cells
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(MDCK; Yool et al., 2002). However, when erythrocytes carrying native AQP1 were tested, tetraethylammonium and tetrapropylammonium showed no significant inhibition even at 10
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mM concentration (Yang et al., 2006). Similarly, stably AQP1-expressing Fisher rat thyroid
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(FRT) epithelial cells were not affected by 1 mM tetraethylammonium (Yang et al., 2006).
Established drug compounds mainly from the various classes of diuretics were tested for AQP inhibition. The diuretic carbonic anhydrase inhibitor acetazolamide that is now used in the treatment of glaucoma and altitude sickness showed significant, dose-dependent, low micromolar inhibition of AQP1 in Xenopus oocytes and in transfected human embryonic kidney 293 cells (HEK293; (Ma et al., 2004; Gao et al., 2006; Seeliger et al., 2013). Again, the inhibiting effect was absent when using erythrocytes and stably AQP1 expressing FRT epithelial cells (Yang et al., 2006). Acetazolamide was further ineffective on purified and reconstituted AQP1 in proteoliposomes; yet, at 1.25 mM reversible, 50% inhibition of reconstituted AQP4 was seen (Tanimura et al., 2009) supporting earlier observations of AQP4 26
ACCEPTED MANUSCRIPT inhibition in Xenopus oocytes (Huber et al., 2007). However, when using transfected FRT cells or glial cells that natively express AQP4, the acetazolamide effect could not be
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reproduced (Yang et al., 2008). At the molecular level this effect may be explained considering that a direct and functional interaction between AQP1 and carbonic anhydrase has
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been recently described (Vilas et al. 2015).
Another approach was to replace the cytosol of Xenopus oocytes with a defined buffer
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(emptied-out oocytes) and application of the loop diuretic furosemide to the intracellular face
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of AQP1. This treatment led to inhibition of AQP1, whereas extracellular furosemide did not affect water permeability (Ozu et al., 2011). A second sulfamyl loop diuretic, bumetanide, significantly reduced the osmotic water flux via AQP4 in Xenopus oocytes and the efficacy
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was increased when the compound was injected (Migliati et al., 2009). A bumetanide
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derivative, AqB013, was even more effective and inhibited AQP1 and AQP4 with an IC50 of
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20 µM when it was applied extracellularly (Migliati et al., 2009).
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Several antiepileptic drugs, topiramate, phenytoin, lamotrigine, as well as triptans, sumatriptan, rizatriptan, were reported to act as inhibitors of AQP4 in the Xenopus oocyte system with double-digit micromolar IC50 (Huber et al., 2009a; Huber et al., 2009b). However, when tested at 100 µM on erythrocytes and stably AQP4-transfected FRT cells no inhibition was observed (Yang et al., 2008). A virtual screening approach using compounds of the ZINC database (Seeliger et al., 2013) yielded several potential extracellular AQP1 inhibitors of which three structurally unrelated compounds inhibited osmotic swelling of AQP1-expressing Xenopus oocytes at low micromolar concentrations. In the same study, the compounds were ineffective in blocking AQP1-mediated water permeability of human erythrocytes.
27
ACCEPTED MANUSCRIPT A likely explanation for the variation in efficacy in different assay system is the number of functional AQP channel proteins present in the respective plasma membrane. Cells exhibiting
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low expression levels should be more sensitive to compounds that bind with low affinity. Care should be taken, that the assay conditions mirror the physiological situation not only
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with respect to the selected AQP isoform but regarding its abundance in the cell membrane, which can be very high (Denker et al., 1988).
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An automated screening assay based on fluorescence changes in calcein-loaded fibroblasts
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and astrocytes that natively express AQP1 and AQP4, respectively, yielded several putative AQP inhibitors (Mola et al., 2009). Re-evaluation within the same study of four hits, NSC164914 and NSC168597 (metallo-compounds; Fig. 4), NSC301460 and NSC670229
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(metal-free; Fig. 4), using erythrocytes and membrane vesicles from transfected AQP4-
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expressing cells confirmed EC50 values in the low micromolar range. While NSC301460 (trychopolyn B) is a fairly complex peptide with a molecular mass of 1188 Da, NSC670229 is
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more drug-like. Recently, the results on AQP1 inhibition were independently reproduced
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using a freeze-thaw assay using yeast cells (To et al., 2015). This latter study produced two additional compounds that inhibited AQP1 water-permeability of human erythrocytes.
With respect to aquaglyceroporins, a small screening approach using a fluorescencequenching assay identified compounds that efficiently inhibited AQP9-mediated glycerol permeability in primary hepatocyte cultures. The IC50 values of these substances were between 0.15 and 4.5 µM (Jelen et al., 2011). Based on these data, putative binding sites were evaluated by point mutations of AQP9 and several more compounds were found to be effective with single-digit micromolar IC50 values in AQP9-expressing Chinese hamster ovary cells (Wacker et al., 2013).
28
ACCEPTED MANUSCRIPT 4.4 Inhibition of the interaction of AQP4 with autoantibodies AQP4 provides extracellular epitopes that are targeted by autoantibodies in the degenerative
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disease neuromyelitis optica (NMO). The aim here is not to modulate AQP4 water permeability but to prevent binding of NMO-IgG antibodies to AQP4. A monoclonal
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antibody, aquaporumab, was generated against extracellular domains of AQP4, which does not affect water permeability but blocks the pathological interaction with NMO-IgG due to steric hindrance as tested in cultured cells, spinal cord slices, and in vivo mouse models of
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NMO (Tradtrantip et al., 2012a). Further, a high-throughput screening was set up to identify
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small molecules that block binding of NMO-IgG to human AQP4. In the assay, recombinant monoclonal NMO-IgG was added to transfected FRT cells that stably express AQP4 in the presence of test compounds (Tradtrantip et al., 2012b). Out of 60,000 compounds the antiviral
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arbidol and the flavonoid tamarixetin were picked up that blocked NMO-IgG binding to
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AQP4 with 5 μM IC50. Statements on the safety and efficacy of both, aquaporumab and
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possibly small NMO-IgG interacting molecules, need to await clinical trials.
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4.5 AQP gene replacement therapy A complication connected to radiation therapy in head and neck cancer is that salivary tissue is destroyed in the process and respective patients suffer severely from dry a mouth leading to ulcers, inflammation, infections, tooth decay, and swallowing difficulties (Baum et al., 2012; Lee et al., 2015). Since the atrophy of laryngeal cells is not treatable in a classical way, gene technological attempts have been made in rat irradiation models to bring AQP1 to expression and restore saliva production (Baum et al., 2012). In a recent human phase I clinical trial, AQP1 cDNA was transferred to eleven individuals by an adenovirus. The study procedures were well tolerated over 42 days. Nevertheless, the risks connected to the use of an adenoviral vector impeded an advancement to phase II trials (Wang et al., 2015). The recent successful
29
ACCEPTED MANUSCRIPT ultrasound-assisted, nonviral gene transfer of AQP1 to an irradiated swine model may open
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up new options for human trials.
5. Conclusions
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The great potential of AQPs to serve as drug targets has been recognized early after their discovery (Beitz & Schultz, 1999). The development of small molecules that specifically modulate AQP function is challenged by spatial restrictions in the protein structure and by the
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high protein abundance in the plasma membranes. Assay systems are demanding with respect
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to instrumentation and handling. The assay outcome depends on the AQP expression level with native cells, such as erythrocytes, being harder to affect. A first set of inhibitors appears to reproducibly inhibit AQPs in independent assay systems indicating that small molecule
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inhibition of AQPs is possible. An alternative approach would be not to target the AQP
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protein directly but to act on intracellular trafficking or signal transduction pathways, such as
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in the case of the AQP2-modulating vaptan class of vasopressin receptor antagonists.
Conflict of Interest Statement The authors declare that there are no conflicts of interest.
30
ACCEPTED MANUSCRIPT Figure legends Figure 1 Physiological situations that require AQP water or glycerol facilitation across cell
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membranes.
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Figure 2 Systems for assaying AQP function. A. Phenotypic assays monitor cell growth or cell death during provision of AQP passing nutrients or toxins, or by exposing the cells to osmotic stress on an hours to days time scale. B. Biophysical assays detect volume changes
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upon osmotic challenges within seconds or subseconds using various physical measures as a
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readout.
Figure 3 Crystal structures of pharmacologically relevant AQPs. AQP1 (PDB# 1FX8), AQP4
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(2D57), and AQP5 (3D9S) are water-specific, PfAQP (3C02) from the malaria parasite
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Plasmodium falciparum is an aquaglyceroporin. The water (blue shading) and glycerol (orange) permeation pathways and vestibules are colored to indicate sites where inhibitor
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binding would directly interfere with AQP function. Diameters of the main constriction site,
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i.e. the aromatic/arginine (ar/R) selectivity filter, are indicated as well as the minimal and maximal width and area of a section through the extracellular vestibule 8 Å above the ar/R region.
Figure 4 Chemical structures of AQP inhibitors. Shown are molecules which were confirmed to be effective by independent investigators using different assay systems. pCMBS – pchloromercuribenzylsulfonate, NSC – numbering scheme according to the Cancer Chemotherapy National Service Center, USA.
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ACCEPTED MANUSCRIPT References Ahmadpour, D., Geijer, C., Tamás, M.J., Lindkvist-Petersson, K. & Hohmann, S. (2014). Yeast reveals unexpected roles and regulatory features of aquaporins and aquaglyceroporins. Biochim Biophys Acta 1840, 1482–1491.
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Almasalmeh, A., Krenc, D., Wu, B. & Beitz, E. (2014). Structural determinants of the hydrogen peroxide permeability of aquaporins. FEBS J 281, 647–656.
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Alsford, S., Eckert, S., Baker, N., Glover, L., Sanchez-Flores, A., Leung, K.F. et al. (2012). High-throughput decoding of antitrypanosomal drug efficacy and resistance. Nature 482, 232–236. Badaut, J., Brunet, J.F. & Regli, L. (2007). Aquaporins in the brain: from aqueduct to "multiduct". Metab Brain Dis 22, 251–263.
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Baker, N., Glover, L., Munday, J.C., Aguinaga Andrés, D., Barrett, M.P., de Koning, H.P. et al. (2012). Aquaglyceroporin 2 controls susceptibility to melarsoprol and pentamidine in African trypanosomes. Proc Natl Acad Sci U.S.A. 109, 10996–11001.
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Baum, B.J., Alevizos, I., Zheng, C., Cotrim, A.P., Liu, S., McCullagh, L. et al. (2012). Early responses to adenoviral-mediated transfer of the aquaporin-1 cDNA for radiation-induced salivary hypofunction. Proc Natl Acad Sci U.S.A. 109, 19403–19407.
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ACCEPTED MANUSCRIPT Binder, D.K., Yao, X., Verkman, A.S. & Manley, G.T. (2006). Increased seizure duration in mice lacking aquaporin-4 water channels. Acta Neurochir Suppl 96, 389–392. Bin, K. & Shi-Peng, Z. (2011). Acetazolamide inhibits aquaporin-1 expression and colon cancer xenograft tumor growth. Hepatogastroenterology 58, 1502–1506.
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Bloch, O., Papadopoulos, M.C., Manley, G.T. & Verkman, A.S. (2005). Aquaporin-4 gene deletion in mice increases focal edema associated with staphylococcal brain abscess. J Neurochem 95, 254–262.
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Borok, Z. & Verkman, A.S. (2002). Lung edema clearance: 20 years of progress: invited review: role of aquaporin water channels in fluid transport in lung and airways. J Appl Physiol 93, 2199–2206.
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Brändén, M., Tabaei, S.R., Fischer, G., Neutze, R. & Höök, F. (2010). Refractive-index-based screening of membrane-protein-mediated transfer across biological membranes. Biophys J 99, 124–133.
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Brooks, H.L., Regan, J.W. & Yool, A.J. (2000). Inhibition of aquaporin-1 water permeability by tetraethylammonium: involvement of the loop E pore region. Mol Pharmacol 57, 1021– 1026. Brown, D. (2003). The ins and outs of aquaporin-2 trafficking. Am J Physiol Renal Physiol 284, 893–901.
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Tradtrantip, L., Zhang, H., Saadoun, S., Phuan, P.W., Lam, C., Papadopoulos, M.C. et al. (2012a). Anti-aquaporin-4 monoclonal antibody blocker therapy for neuromyelitis optica. Ann Neurol 71, 314–322. Tradtrantip, L., Zhang, H., Anderson, M.O., Saadoun, S., Phuan, P.W., Papadopoulos, M.C. et al. (2012b). Small-molecule inhibitors of NMO-IgG binding to aquaporin-4 reduce astrocyte cytotoxicity in neuromyelitis optica. FASEB J 26, 2197–2208. Tsubota, K., Hirai, S., King, L.S., Agre, P. & Ishida, N. (2001). Defective cellular trafficking of lacrimal gland aquaporin-5 in Sjögren's syndrome. Lancet 357, 688–689. Uzcategui, N.L., Szallies, A., Pavlovic-Djuranovic, S., Palmada, M., Figarella, K., Boehmer, C. et al. (2004). Cloning, heterologous expression, and characterization of three aquaglyceroporins from Trypanosoma brucei. J Biol Chem 279, 42669–42676. Vacca, A., Frigeri, A., Ribatti, D., Nicchia, G.P., Nico, B., Ria, R. et al. (2001). Microvessel overexpression of aquaporin 1 parallels bone marrow angiogenesis in patients with active multiple myeloma. Br J Haematol 113, 415–421. Verkman, A.S. (2003). Role of aquaporin water channels in eye function. Exp Eye Res 76, 137–143. Verkman, A.S. (2005). More than just water channels: unexpected cellular roles of aquaporins. J Cell Sci 118, 3225–3232.
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ACCEPTED MANUSCRIPT Verkman, A.S. (2007). Role of aquaporins in lung liquid physiology. Respir Physiol Neurobiol 159, 324–330. Verkman, A.S. (2008). A cautionary note on cosmetics containing ingredients that increase aquaporin-3 expression. Exp Dermatol 17, 871–872.
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Verkman, A.S., Anderson, M.O. & Papadopoulos, M.C. (2014). Aquaporins: important but elusive drug targets. Nat Rev Drug Discov 13, 259–277.
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Vilas, G., Krishnan. D., Loganathan, S.K., Malhotra, D., Liu, L., Beggs, M.R., Gena, P., Calamita, G., Jung, M., Zimmermann, R., Tamma, G., Casey, J.R. & Alexander, R.T. (2015). Increased water flux induced by an aquaporin-1/carbonic anhydrase II interaction. Mol Biol Cell 26, 1106-1118. Villabona, C. (2010). Antagonistas del receptor de vasopresina: los vaptanes. Endocrinol Nutr 57 Suppl 2, 41–52.
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Wacker, S.J., Aponte-Santamaría, C., Kjellbom, P., Nielsen, S., de Groot, B.L. & Rützler, M. (2013). The identification of novel, high affinity AQP9 inhibitors in an intracellular binding site. Mol Membr Biol 30, 246–260.
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Wang, F., Feng, X.C., Li, Y.M., Yang, H. & Ma, T.H. (2006). Aquaporins as potential drug targets. Acta Pharmacol Sin 27, 395–401.
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Wang, Z., Zourelias, L., Wu, C., Edwards, P.C., Trombetta, M. & Passineau, M.J. (2015). Ultrasound-assisted nonviral gene transfer of AQP1 to the irradiated minipig parotid gland restores fluid secretion. Gene Ther doi: 10.1038/gt.2015.36. [Epub ahead of print].
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Wree, D., Wu, B., Zeuthen, T. & Beitz, E. (2011). Requirement for asparagine in the aquaporin NPA sequence signature motifs for cation exclusion. FEBS J 278, 740–748. Wu, B. & Beitz, E. (2007). Aquaporins with selectivity for unconventional permeants. Cell Mol Life Sci 64, 2413–2421.
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Wu, B., Altmann, K., Barzel, I., Krehan, S. & Beitz, E. (2008). A yeast-based phenotypic screen for aquaporin inhibitors. Pflugers Arch 456, 717–720.
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Wu, B., Steinbronn, C., Alsterfjord, M., Zeuthen, T. & Beitz, E. (2009). Concerted action of two cation filters in the aquaporin water channel. EMBO J 28, 2188–2194. Wu, B., Song, J. & Beitz, E. (2010). Novel channel enzyme fusion proteins confer arsenate resistance. J Biol Chem 285, 40081–40087. Yang, B., Gillespie, A., Carlson, E.J., Epstein, C.J. & Verkman, A.S. (2001). Neonatal mortality in an aquaporin-2 knock-in mouse model of recessive nephrogenic diabetes insipidus. J Biol Chem 276, 2775–2779. Yang, B., Kim, J.K. & Verkman, A.S. (2006). Comparative efficacy of HgCl2 with candidate aquaporin-1 inhibitors DMSO, gold, TEA+ and acetazolamide. FEBS Lett 580, 6679–6684. Yang, B., Zhang, H. & Verkman, A.S. (2008). Lack of aquaporin-4 water transport inhibition by antiepileptics and arylsulfonamides. Bioorg Med Chem 16, 7489–7493. Yang, B., Zhao, D. & Verkman, A.S. (2009). Hsp90 inhibitor partially corrects nephrogenic diabetes insipidus in a conditional knock-in mouse model of aquaporin-2 mutation. FASEB J 23, 503–512. Yasui, M., Hazama, A., Kwon, T.H., Nielsen, S., Guggino, W.B. & Agre, P. (1999). Rapid gating and anion permeability of an intracellular aquaporin. Nature 402, 184–187.
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ACCEPTED MANUSCRIPT Yool, A.J., Brokl, O.H., Pannabecker, T.L., Dantzler, W.H. & Stamer, W.D. (2002). Tetraethylammonium block of water flux in Aquaporin-1 channels expressed in kidney thin limbs of Henle's loop and a kidney-derived cell line. BMC Physiol 2, 4.
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Yukutake, Y., Tsuji, S., Hirano, Y., Adachi, T., Takahashi, T., Fujihara, K. et al. (2008). Mercury chloride decreases the water permeability of aquaporin-4-reconstituted proteoliposomes. Biol Cell 100, 355–363.
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Zelenina, M., Bondar, A.A., Zelenin, S. & Aperia, A. (2003). Nickel and extracellular acidification inhibit the water permeability of human aquaporin-3 in lung epithelial cells. J Biol Chem 278, 30037–30043. Zelenina, M., Tritto, S., Bondar, A.A., Zelenin, S. & Aperia, A. (2004). Copper inhibits the water and glycerol permeability of aquaporin-3. J Biol Chem 279, 51939–51943.
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Zeuthen, T., Wu, B., Pavlovic-Djuranovic, S., Holm, L.M., Uzcategui, N.L., Duszenko, M. et al. (2006). Ammonia permeability of the aquaglyceroporins from Plasmodium falciparum, Toxoplasma gondii and Trypansoma brucei. Mol Microbiol 61, 1598–1608.
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Zhang, D., Vetrivel, L. & Verkman, A.S. (2002). Aquaporin deletion in mice reduces intraocular pressure and aqueous fluid production. J Gen Physiol 119, 561–569. Zhang, H. & Verkman, A.S. (2010). Aquaporin-1 tunes pain perception by interaction with Na(v)1.8 Na+ channels in dorsal root ganglion neurons. J Biol Chem 285, 5896–5906.
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Zhang, R., van Hoek, A.N., Biwersi, J. & Verkman, A.S. (1993). A point mutation at cysteine 189 blocks the water permeability of rat kidney water channel CHIP28k. Biochemistry 32, 2938–2941.
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Zhang, Z., Chen, Z., Song, Y., Zhang, P., Hu, J. & Bai, C. (2010). Expression of aquaporin 5 increases proliferation and metastasis potential of lung cancer. J Pathol 221, 210–220.
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Figure 1
44
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ACCEPTED MANUSCRIPT
AC
CE
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Figure 2
45
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ACCEPTED MANUSCRIPT
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Figure 3
46
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Figure 4
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ACCEPTED MANUSCRIPT Table 1. incubation time (min)
extent of inhibitio n
300 and 3000
5
significa nt
300
0 (only in assay solution) 5
significa nt
300
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30 2.5
significa nt 68 %
tetraethylammoni um
acetazolamide (carboanhydrase inhibitor)
2.5
75 %
10
not significa nt
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HAuCl3
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100
100
3.9
6
ED
15
silver sulfadiazine
referenc e
Xenopus oocytes
Preston et al., 1992 Zhang et al., 1993
Xenopus oocytes
10
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(50 mg/ml)
test system
Xenopus oocytes
significa nt
15 pCMB-conjugated dextran AgNO3
IC50 (μM )
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AQP1 HgCl2
test conc. (μM)
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test compound
15
1.24
significa nt
15
46 %
1000
240
34.2 %
10000
240
not significa nt
1000
15
10
15
1.4
81 %
15
5.5
48
mouse and human erythrocytes human erythrocytes human erythrocytes
14
15
mouse and human erythrocytes human erythrocytes human erythrocytes
mouse and human erythrocytes Xenopus oocytes Xenopus oocytes AQP1-MDCK cells mouse and human erythrocytes AQP1-FRT cells Xenopus oocytes Xenopus oocytes
Preston et al., 1993 Yang et al., 2006 Zhang et al., 1993 Niemietz & Tyerman , 2002 Yang 2006 Niemietz & Tyerman , 2002 Martins et al., 2012 Yang et al., 2006 Brooks et al., 2000 Detmers et al., 2006 Yool et al., 2002 Yang et al., 2006
Ma et al., 2004 Seeliger et al., 2013
ACCEPTED MANUSCRIPT 30
39 %
AQP1-HEK293
2000
240
not significa nt
1000
15
1250
30
mouse and human erythrocytes AQP1-FRT cells AQP1proteoliposom es Xenopus oocytes human erythrocytes
15 10 – 20
compound 2
15 10 – 20
compound 3
15
5 and 60 60 – 120
significa nt
15
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NSC670229
Ozu et al., 2011
20
5
significa nt
Xenopus oocytes
15
40 %
significa nt 49 significa nt 28
20
tetraethylammoni um
emptied-out Xenopus oocytes Xenopus oocytes
27
15
300
Seeliger et al., 2013
significa nt
15
20
AQP2 HgCl2
Xenopus oocytes human erythrocytes
significa nt
15
NSC301460
17.5
rat erythrocytes yeast freezethaw assay rat erythrocytes yeast freezethaw assay rat erythrocytes yeast freezethaw assay rat erythrocytes yeast freezethaw assay
20
NSC168597
Seeliger et al., 2013
40
20
49
Tanimur a et al., 2009 Seeliger et al., 2013
Xenopus oocytes human erythrocytes
not significa nt ca. 66 %
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10 (intracellula r) 20
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NSC164914
10 – 20
Gao et al., 2006 Yang et al., 2006
17.0
not significa nt
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4
not significa nt
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20
AqB013
8.1
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20
furosemide (loop diuretic)
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compound 1
not significa nt
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100
6.2
Xenopus oocytes
Migliati et al., 2009 Mola et al., 2009 To et al., 2015 Mola et al., 2009 To et al., 2015 Mola et al., 2009 To et al., 2015 Mola et al., 2009 To et al., 2015 Fushimi et al., 1993 Detmers et al., 2006
ACCEPTED MANUSCRIPT
Aubipy
30
AubipyMe
30
AubipyNH2
30
Auterpy
30
Cuphen
30
AQP4 HgCl2
CE
1250
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acetazolamide (carboanhydrase inhibitor)
50 %
15
57 %
5
53.3 %
120
80 % at 20 µM not significant
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tetraethylammoni um
5
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5
human erythrocytes
16 .6
human erythrocytes
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30
0. 8
2. 3
human erythrocytes
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Audien
89 % (glycerol) at 100 µM 79 % (glycerol) at 50 µM 92,7 % (glycerol) at 10 µM 93,1 % (glycerol) at 10 µM 80 % (glycerol) at 10 µM 89 % (glycerol) at 10 µM 89 % (glycerol) at 1000 µM
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10
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AQP3 Auphen
1. 0
human erythrocytes
2. 9
human erythrocytes
1. 0
human erythrocytes
81 .9
human erythrocytes
9. 8
AQP4proteoliposom es Xenopus oocytes
0. 9
AQP4 proteoliposom es Xenopus oocytes AQP4-FRT cell membrane vesicles AQP4-FRT cell monolayers native glial cells Xenopus oocytes
100
15
5 (intracellula r)
120 – 240
significant
2-(nicotinamido)1,3,4-thiadiazole
120
67 % at 20 μM
3. 1
Xenopus oocytes
sumatriptan (H1-recept. agonist; anti-migraine)
120
54 % at 20 μM
11
Xenopus oocytes
15
not significant
furosemide (loop diuretic)
100
50
AQP4-FRT cell membrane vesicles AQP4-FRT cell
Martins et al., 2012 Martins et al., 2012 Martins et al., 2013 Martins et al., 2013 Martins et al., 2013 Martins et al., 2013 Martins et al., 2013 Yukutak e et al., 2008 Detmers et al., 2006 Tanimur a et al., 2009 Huber et al., 2007 Yang et al., 2008
Migliati et al., 2009 Huber et al., 2009a Huber et al., 2009a Yang et al., 2008
ACCEPTED MANUSCRIPT 52 % at 20 μM
20
120
68 %
100
15
not significant
20
120
23 %
120
67 % at 20 μM 75 % at 100 μM not significant
10
48 % at 20 μM 48 % at 100 μM not significant
58 % at 20 μM 60 % at 100 μM not significant
topiramate (antiepileptic drug)
15
ED 120
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zonisamide (antiepileptic drug)
15
AC
CE
100
phenytoin (antiepileptic drug)
oxcarbazepine (antiepileptic drug) lamotrigine (antiepileptic drug)
120
100
15
20
120
33 %
120
54 % at 20 μM 64 % 100 μM not significant
100
monolayers Xenopus oocytes
15
51
Huber et al., 2009a Huber et al., 2007 Yang et al., 2008
Yang et al., 2008
3. 3
AQP4-FRT cell membrane vesicles AQP4-FRT cell monolayers native glial cells Xenopus oocytes
Yang et al., 2008
9. 8
AQP4-FRT cell membrane vesicles AQP4-FRT cell monolayers Xenopus oocytes AQP4-FRT cell membrane vesicles AQP4-FRT cell monolayers Xenopus oocytes
Yang et al., 2008
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Xenopus oocytes AQP4-FRT cell membrane vesicles AQP4-FRT cell monolayers Xenopus oocytes Xenopus oocytes
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100
2. 9
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compound 4
120
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rizatriptan (H1-recept. agonist; anti-migraine) 6-ethoxybenzothiazole-2sulfonamide
8. 1
Xenopus oocytes AQP4-FRT cell membrane vesicles AQP4-FRT cell
Huber et al., 2007 Huber et al., 2009b
Huber et al., 2009b
Huber et al., 2009b
Huber et al., 2009b Huber et al., 2009b Yang et al., 2008
ACCEPTED MANUSCRIPT 120
40 %
carbamazepine10,11-epoxide (antiepileptic drug)
20
120
40 %
bumetanide (loop diuretic)
100 (extracellula r) 50 (intracellula r)
60 – 120 (extracellula r) 120 – 240 (intracellula r) 60 – 120
significant
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AqB013
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AQP9 RF03176 S14838
HTS13286
1.3 1.5
0.15 410
AC
CE
ID1 – ID6
20
3.0
PT
HTS13772
Xenopus oocytes
4.5
ED
CD05595
monolayers Xenopus oocytes
PT
20
52
Huber et al., 2009b Huber et al., 2009b
Xenopus oocytes
Migliati et al., 2009
Xenopus oocytes
Migliati et al., 2009
AQP9-CHO cells AQP9-CHO cells AQP9-CHO cells AQP9-CHO cells AQP9-CHO cells AQP9-CHO cells
Jelen et al., 2011 Jelen et al., 2011 Jelen et al., 2011 Jelen et al., 2011 Jelen et al., 2011 Wacker et al., 2013
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valproic acid (valproic acid)
S0163-7258(15)00160-6 doi: 10.1016/j.pharmthera.2015.08.002 JPT 6806
To appear in:
Pharmacology and Therapeutics
Please cite this article as: Beitz, E., Golldack, A., Rothert, M. & von B¨ ulow, J., Challenges and achievements in the therapeutic modulation of aquaporin functionality, Pharmacology and Therapeutics (2015), doi: 10.1016/j.pharmthera.2015.08.002
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ACCEPTED MANUSCRIPT Invited Review (edt. Enno Klussmann)
Rev. August 2, 2015
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P & T #22780
Challenges and achievements in the therapeutic modulation of aquaporin functionality
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Eric Beitz, André Golldack, Monja Rothert, Julia von Bülow
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Pharmaceutical and Medicinal Chemistry, University of Kiel, Germany
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Correspondence address:
Eric Beitz Pharmaceutical and Medicinal Chemistry University of Kiel Gutenbergstrasse 76 24118 Kiel, Germany Tel. Fax E-mail:
++49 431 880 1809 ++49 431 880 1352 [email protected]
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ACCEPTED MANUSCRIPT Abstract Aquaporin water and solute channels (AQP) have basic physiological functions throughout
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the human body. AQP-facilitated water permeability across cell membranes is required for rapid reabsorption of water from pre-urine in the kidneys and for sustained near isosmolar
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water fluxes e.g. in the brain, eyes, inner ear, and lungs. Cellular water permeability is further connected to cell motility. AQPs of the aquaglyceroporin subfamily are necessary for lipid degradation in adipocytes and glycerol uptake into the liver, as well as for skin moistening.
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Modulation of AQP function is desirable in several pathophysiological situations, such as
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nephrogenic diabetes insipidus, Sjögren’s syndrome, Menière’s disease, heart failure, or tumors to name a few. Attempts to design or to find effective small molecule AQP inhibitors have yielded only a few hits. Challenges reside in the high copy number of AQP proteins in
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the cell membranes, and spatial restrictions in the protein structure. This review gives an
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overview on selected physiological and pathophysiological conditions in which modulation of
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AQP inhibitors.
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AQP functions appears beneficial and discusses first achievements in the search of drug-like
Keywords: Aquaporin, water, glycerol, volume regulation, inhibition, drug development
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ACCEPTED MANUSCRIPT Table of Contents 1. Introduction
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2. Selected physiological and pathophysiological roles of AQPs 2.1 Kidneys, inner ear
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2.2 Eye, salivary glands 2.3 Central nervous system 2.4 Lungs
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2.5 Tumors
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2.6 Immune system, hematocytes, parasite infections 2.7 Fat metabolism
3. Assay systems for AQP function and inhibition
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3.1 Phenotypic AQP assays
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3.2 Biophysical AQP assays
3.3 High-throughput AQP assays
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4. Towards therapeutic modulation of AQP function
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4.1 AQP protein structures as drug target 4.2 AQP inhibition by transition metals 4.3 AQP inhibition by small organic molecules 4.4 Inhibition of the interaction of AQP4 with autoantibodies 4.5 AQP gene replacement therapy 5. Conclusion
Abbreviations: AQP – aquaporin, NMO – neuromyelitis optica
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ACCEPTED MANUSCRIPT 1. Introduction A survey of the currently used pharmacological agents and classification of their respective
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drug targets puts a figure on the large predominance of compounds that address receptor molecules (44% of all human drug targets; Rask-Andersen et al., 2011). Within the class of
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receptor drug targets almost half represent G-protein coupled receptors and one fifth are ligand-gated ion channel receptors, followed by tyrosine-kinase receptors. The next large target group contains various enzymes (29% of the drug targets) with a major focus on lipid
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mediator-producing oxidoreductases, such as the cyclooxygenases, COX, i.e. the targets of
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nonsteroidal anti-inflammatory drugs. The drug target class of transport and channel proteins ranks third (15% of all drug targets). Here, mainly ion channels of the heart and circular system are modulated in their function to treat arrhythmia and hypertension as well as
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neurotransmitter transporters of the brain for neurological disorders. The new anti-diabetic
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class of gliflozins, that inhibit the renal sodium-dependent glucose transporter, SGLT2, represents one of the very few cases in which high-level transport of a nutrient at millimolar
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concentrations is targeted.
This brief overview shows that the great majority of drugs interfere fairly directly with signal transduction, be it at the receptor, the small-molecule transmitter, or the action potential level. The advantages are obvious: the drugs compete with very low transmitter concentrations, which can be as low as in the femtomolar range, and the target proteins exhibit explicit binding sites, which allow for high-affinity interactions. Both are prerequisites for optimizing drug compounds towards low-dose application and specificity of action.
The situation of transmembrane water transport facilitated by one of the thirteen different human aquaporin channel proteins (AQP0-12) is vastly opposite in both, substrate concentration and affinity aspects. Water represents the most abundant molecule in the human 4
ACCEPTED MANUSCRIPT body (about 60% of the total body mass) and its concentration in the body fluids is 55 molar, i.e. 10,000 times higher than that of the most important energy carrier molecule glucose. With
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respect to substrate affinity, AQP proteins appear inconspicuous to passing water molecules by mimicking the hydrogen bond situation and binding energy of the aqueous bulk. Thus, the
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energy barrier for water permeation through the AQP channel following an osmotic gradient is hardly higher than diffusion in free solution. Structure-wise, AQPs are rigid proteins and exhibit little thermal fluctuations of the amino acid residues in the channel region in order to
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maintain a 20 Å long and very narrow channel pathway of only 2-4 Å in diameter open for
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water (orthodox AQPs; Murata et al., 2000) or small, uncharged solutes, mainly glycerol and chemically resembling compounds (aquaglyceroporins; Fu et al., 2000), posing major space limitations for putative inhibitor molecules. On top of that, AQPs tend to populate the cell
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membranes in large numbers, i.e. the membrane of a single erythrocyte contains about
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200,000 AQP copies (Solomon et al., 1983; Denker et al., 1988).
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Despite the challenges due to the AQP structure and protein abundance, it appears worthwhile
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to search for small molecule modulators due to the many and central roles AQPs play in physiology and pathophysiology. A recent review by Verkman et al. (2014) provides a comprehensive and excellent overview on AQP-related disorders and pharmacological intervention attempts. In this paper, we will focus – after going through physiology and AQPrelated therapeutic possibilities – on options for compound screening, and the protein structural and chemical aspects of AQP modulator design.
2. Selected physiological roles of AQPs and options for modulation Besides water and glycerol, AQPs facilitate permeation, dependent on the isoform, of various other physiological molecules across cell membranes: ammonia, carbon dioxide, urea, hydrogen peroxide, and methylglyoxal (Wu & Beitz, 2007). Potential physiological roles of 5
ACCEPTED MANUSCRIPT AQPs are, thus, in waste metabolite elimination (ammonia, urea, methylglyoxal), cellular gas exchange (carbon dioxide), and oxidative stress relief and/or signal molecule transport
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(hydrogen peroxide). Such AQP functions, if physiologically relevant, have not been attributed to diseases, yet. Hence, in the following, we will summarize data on the more
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classical roles of AQPs that are mainly related to water and glycerol transmembrane transport and in which pharmacological modulation is considered beneficial.
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Lipid bilayers are permeable for water; yet, due to the lipophilic membrane core, osmotic
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diffusion rates are low and considerable activation energy (> 10 kcal mol–1) is required. In the presence of AQP water channels, transmembrane water permeability increases by one to two orders of magnitude and the energetic cost is lowered to that of breaking two to three
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hydrogen bonds (< 5 kcal mol–1; Preston et al., 1992). Two situations call for AQP facilitated
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transmembrane water transport (Fig. 1): a) rapid, high-volume transport (kidneys), and b) sustained water transport at small, near-isosmotic gradients (slow fluid exchange, secretory
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glands, cell motility). Transmembrane transport of glycerol along a chemical gradient is
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relevant in skin moistening and in the Cori cycle, i.e. glycerol release from adipocytes during lipolysis and uptake of glycerol by the liver for gluconeogenesis. We will discuss water transport in the kidneys and the inner ear in one section because in both cases regulation is via vasopressin; thereafter, we address the situation in the eye and surrounding secretory tissues together with water secreting salivary glands.
2.1 Kidneys, inner ear The water transport capacity of the kidneys is unparalleled in the human body. It is driven by a steep osmotic gradient due to the active transport of salt reaching concentrations up to four times higher than in normal tissue. More than 150 liters of blood are filtered by the nephrons per day, equaling 100 ml min–1 or 1.7 ml s–1. This way, hydrophilic, potentially toxic 6
ACCEPTED MANUSCRIPT substances, such as waste metabolites and xenobiotics, are cleared from the body. At the same time, the kidneys regulate the water, salt, and pH homeostasis of the organism. To maintain
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the water-balance, about 99% of the filtered pre-urine water is being reabsorbed. The proximal tubule and the descending thin limb of the Henle loop continuously take up the
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major volume, whereas the remaining, approximately 20% of the kidney filtrate is used to adjust homeostasis via the action of the hormone vasopressin that acts on the water
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permeability of the collecting duct endothelia.
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The discovery of the AQPs eventually provided the molecular basis for the highly water permeable kidney sections. In total, eight AQPs have been localized in different segments of
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the nephron.
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AQP1 is the predominant water channel in the apical and basolateral membranes of the brush border cells of the proximal tubules, in the loop of Henle, as well as in the endothelium of the
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descending vasa recta (Nielsen et al., 1993b). Accordingly, AQP1-null mice suffer from a
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major reduction in water reabsorption in the proximal tubules (Ma et al., 1998; Schnermann et al., 1998; Chou et al., 1999; Pallone et al., 2000). HgCl2 and organo-mercurials are known AQP1 inhibitors that bind to a cysteine residue (Cys189 in human AQP1) close to the extracellular pore entry. Such mercurials were used as diuretics until the 1950s (Nielsen et al., 1999) and AQP1 is one putative site of action. Due to the highly unspecific nature of cysteine-modification by mercurials it is very likely, though, that other protein components of the kidney water reabsorption system, e.g. ion channels, were hit as well. Likewise, other AQPs carrying a cysteine in the pore region are inhibited by mercurials. The co-incidence that AQP1 is additionally present in red blood cell membranes and exposes the Colton blood group epitope, led to the identification of women lacking AQP1 altogether. Their urine concentration defect became evident only when water drinking was restricted whereas with 7
ACCEPTED MANUSCRIPT unrestricted fluid uptake no polyuria was noticeable suggesting compensation by other AQPs
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or unknown mechanisms present in the human kidney (King et al., 2001).
AQP2 is a second major kidney water channel present in the principal cells of the collecting
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duct (Fushimi et al., 1994) where it acts as an indirect drug target of the new class of vaptans, i.e. vasopressin type 2 receptor antagonists (Villabona, 2010). Under basal conditions, AQP2 is stored in perinuclear vesicles, which translocate, upon vasopressin-elicited cAMP signaling
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and phosphorylation of AQP2 in the C-terminal region by protein kinase A, to the apical
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plasma membrane (Nielsen et al., 1993a; Brown, 2003; Sasaki, 2012). Incorporation of AQP2 into the plasma membrane increases water permeability and, thus, reabsorption of pre-urine. Functional defects in the vasopressin type 2 receptor or in AQP2 result in a daily production
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of up to 20 liters of a dilute urine, i.e. nephrogenic diabetes insipidus (Deen et al., 1994;
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Brown, 2003; Kamsteeg et al., 2003; Nguyen et al., 2003). Mouse models lacking kidney AQP2 (Rojek et al., 2007) or expressing an intracellularly misrouted AQP2 variant
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(Thr126Met; Yang et al., 2001) confirmed the role of AQP2 in vasopressin-dependent urine
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volume regulation. The phenotype of the latter could be partially corrected by an inhibitor of heat-shock protein 90 (Yang et al., 2009). Nephropathies induced by lithium, hypokalemia, or by cisplatin treatment can manifest as forms of nephrogenic diabetes insipidus and appear to be associated with decreased expression of AQP2 (Nielsen et al., 2002). Vaptans as inhibitors of the vasopressin/AQP2-dependent water reabsorption act as aquaretics (increasing the secreted water volume) rather than as diuretics (increasing salt and water secretion; Villabona, 2010). Vaptans are used to treat clinical conditions associated with water retention, such as in congestive heart failure, or liver cirrhosis (Schrier et al., 1998; Nielsen et al., 2002), as well as euvolemic and hypervolemic hyponatremia (Izumi et al., 2014). Specific direct inhibitors of kidney AQP1 or AQP2 should be applicable for the same indications.
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ACCEPTED MANUSCRIPT Vaptans have further been successfully used to treat an animal model of surgically induced inner ear hydrops (Takeda et al., 2003). The molecular setup of the inner ear endolymph
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volume regulation system appears to be highly similar to that of the kidney and includes the vasopressin type 2 receptor and AQP2 (Kumagami et al., 1998; Beitz et al., 1999).
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Endolymph hydrops, i.e. an overpressure in the endolymph compartment harboring the sensory cells for hearing and balance, is responsible for the symptoms of Menière’s disease,
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hearing loss and severe vertigo attacks.
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Of the remaining six kidney AQPs, AQP3 and AQP4 co-localize with AQP2 in the collecting duct but are constitutively present in the basolateral membranes of the principal cells where they form the exit pathways for reabsorbed water into the tissue (Nielsen et al., 2002). AQP3
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knockout mice exhibit a polyuric and polydipsic phenotype comparable to that of AQP1 (Ma
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et al., 2000). AQP4 accounts for 75 % of basolateral water transport in mice, yet deletion of the AQP4 gene reduced the ability to concentrate urine only moderately (Ma et al., 1997).
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Recently, AQP5 was localized in type B intercalated cells of the collecting duct (Porcino et al.
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2011). Since the AQP5 is present in the apical membrane and the basolateral membrane lacks AQPs it was speculated that the cells may act in osmosensing. AQP6 is expressed in acidsecreting intercalated cells of collecting ducts and co-localizes intracellularly with the vacuolar H+-ATPase (Yasui et al., 1999; Ikeda et al., 2002). AQP6 is permeable to anions, such as nitrite and chloride, and is gated by pH suggesting a role in vesicle acidification; however, the phenotype of an AQP6 knock-out mouse is still elusive. AQP7 and AQP8 are expressed in the proximal tubule of the nephron (Nielsen et al., 2002), but a renal function has not been attributed to these isoforms, yet. The knockout of AQP11 in mice, however, leads to a severe and lethal phenotype of massive vacuolization and cyst formation in the proximal tubules (Morishita et al., 2005); the animals die within two months after birth. AQP11 is localized intracellularly in the endoplasmic reticulum from which the vacuolization process is 9
ACCEPTED MANUSCRIPT initiated when AQP11 is absent (Okada et al., 2008). The resemblance of cyst formation in the knockout mice and in polycystic kidney disease models may help elucidating the
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underlying molecular mechanisms leading to the disease.
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2.2 Eye, salivary glands
The sensory cells of the eye’s retina are situated in an enclosed fluid-filled chamber, which requires, similar to the inner ear, careful volume regulation by AQPs to adjust the intraocular
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pressure (Frigeri et al., 1995). AQP1 and AQP4 are expressed in the iris and the ciliar
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epithelium and are thought to carry the major portion of chamber water production (Zhang et al., 2002). Increased intraocular pressure, glaucoma, poses stress to the cells of the retina and the optic nerve (Huber et al., 2012). There are pharmacological agents for the treatment of
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both, wide-angle and narrow-angle glaucoma, and inhibitors of AQP1 and AQP4 may become
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alternatives to adjust water regulation in the eye.
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AQP water channels are further central for the maintenance of cornea and lens transparency
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(Verkman, 2003), e.g. corneal thickness is decreased in AQP1-null mice (Huber et al., 2012). This example and the fact that AQP1 is generally the most widely distributed AQP throughout the human body should be considered with respect to putative side effects when thinking about systemic inhibition of AQP1. AQP0 of the eye lens was found to have low waterpermeability, its main function appears to be structural and to reside in the parallel fixation of cells by cell-cell contacts (Mulders et al., 1995). Mutations in AQP0 have been associated with human congenital cataract (Berry et al., 2000; Geyer et al., 2006). Correction of such structural effects by pharmacologically means seems difficult; yet, the accessibility of the cornea and lens may permit gene therapeutic approaches to replace defect AQP0 in the future.
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ACCEPTED MANUSCRIPT AQP3, found in the conjunctiva, and AQP5, in lacrimal glands, are required for the secretion of eye surface liquids (King et al., 2004). Respective agonists may be beneficial in treating
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dry eye syndrome. Patients with Sjøgren´s syndrome suffering from dry eyes and mouth have been identified to carry mutations in AQP5 that lead to intracellular misrouting of the water
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channel in the lacrimal and salivary glands (Steinfeld et al., 2001; Tsubota et al., 2001). A causative therapy would have to re-establish proper AQP5 levels and localization to the apical
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membranes in the affected cells.
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2.3 Central nervous system
Changes in the volume of the cerebrospinal fluid directly pose stress on the brain cells whereas changes in the ionic composition of the fluid can alter neuronal signaling. Seven
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AQPs were amplified from cDNA of mammalian brain cells; however, on the protein level,
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only three AQPs could be confirmed of which AQP1 and AQP4 predominate (Badaut et al., 2007), whereas AQP9 protein levels are low and knockout mice do not exhibit a cerebral
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phenotype. AQP1 is found in the choroid plexus epithelium (Nielsen et al., 1993c) where it
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facilitates secretion of the cerebrospinal fluid and regulation of the intracranial pressure (Oshio et al., 2005); inhibition of AQP1 may be an option for the treatment of hydrocephalis (Tait et al., 2008). AQP1 is also present in neuronal cells, such as nociceptive C-fibers (Oshio et al., 2004; Oshio et al., 2006; Shields et al., 2007) and dorsal root ganglion neurons (Zhang & Verkman, 2010). An involvement of AQP1 water permeability in pain sensing is discussed and AQP1 inhibitors may even turn out as a novel principle in analgesic therapy.
AQP4 is the most abundant AQP in the human brain and is predominantly found in the basolateral membrane of ependymal cells, and in astrocytes interfacing the cerebrospinal fluid compartment and the blood (Nielsen et al., 1997; Rash et al., 1998). AQP4 knockout mice were better protected from brain edema induced by water intoxication or cerebral ischemic 11
ACCEPTED MANUSCRIPT injury than wild-type animals and exhibited less neurological damage and lower rates of mortality suggesting a putative role of AQP4 inhibitors in the acute treatment of such edema
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(Manley et al., 2000; Papadopoulos & Verkman, 2005; Haj-Yasein et al., 2011; Katada et al., 2014). Oppositely, AQP4 appears to be responsible for the volume clearance in vasogenic
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brain edema that is caused by increased permeability of the blood brain barrier (Papadopoulos et al., 2004). AQP4 null mice suffered from increased intracranial pressure giving a worsened clinical outcome compared to wild-type mice, when vasogenic edema was induced by
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continuous intracerebral fluid infusion, freeze-injury, an intraparenchymal bacterial abscess,
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or brain tumors (Bloch et al., 2005; Papadopoulos et al., 2004; Papadopoulos & Verkman, 2005). Accordingly, inhibition of AQP4 would require secure diagnosis of the underlying cause of the edema. Besides water regulation, neuronal activity of AQP4 null mice was
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affected as seen by a higher seizure threshold and prolonged seizure duration, which may be
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beneficial in epilepsy therapy (Binder et al., 2006); at the same time, mice lacking AQP4 are deaf, again pointing to severe potential side effects of AQP4 inhibitors (Li & Verkman,
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2001).
AQP4 is also associated with neuromyelitis optica (NMO), an inflammatory autoimmune disease of the optic nerve and the spinal cord. 60-90% of the patients generate autoantibodies that are directed against AQP4 and cause complement and immune cell-mediated damage to astrocytes (Jarius & Wildemann, 2010; Lennon et al., 2005). Current developments aim at the inhibition of the interaction of AQP4 and the autoantibodies and are discussed below in section 4.4.
2.4 Lungs Water transport across epithelial and endothelial barriers in bronchopulmonary tissues occurs during airway hydration, alveolar fluid transport, and submucosal gland secretion (Borok & 12
ACCEPTED MANUSCRIPT Verkman, 2002). AQP1, AQP3, AQP4 and AQP5 are expressed in the lung and are candidates for facilitating respective water transport (Verkman, 2007). Human AQP1-null
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individuals showed thickening of the airway walls, i.e. signs of peribronchiolar edema, after ingestion of large quantities of fluid (King et al., 2002). In mouse studies, however, deletion
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of genes encoding AQP1, AQP3, or AQP4 did not produce a lung phenotype. Solely, AQP5null mice showed impaired fluid secretion by airway submucosal glands and subsequent changes in the composition, viscosity, and volume of the airway surface liquid (Song &
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Verkman, 2001). Therapeutic modulation of AQP5 on the transcriptional or functional level,
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thus, could be meaningful in cystic fibrosis (Verkman, 2007).
2.5 Tumors
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Different AQPs have been found in various tumors and related cell types (Saadoun et al.,
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2002b; Saadoun et al., 2002a). Their role apparently does not solely reside in keeping up water balance as laid out above, but in bringing about cellular functions regarding migration,
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proliferation, and adhesion (Verkman et al., 2014), which are prerequisites for tumor growth, angiogenesis, metastasis, and tissue infiltration. The field of AQP-related tumor biology is
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rapidly expanding; hence, we will highlight some examples and refer to specific recent reviews for further reading (Papadopoulos & Saadoun, 2014; Ribatti et al., 2014; Verkman et al., 2014).
Research in the field was ignited by the notion that in AQP1-null mice subcutaneously or intracranially implanted tumors grew markedly slower, as did breast tumors and lung metastases (Saadoun et al., 2005; Esteva-Font et al., 2014). The growth defect was due to increased necrosis and reduced vascularisation. Typically, AQP1 is found in respective microvessels (Endo et al., 1999; Vacca et al., 2001). Primary cultures of vessel endothelial cells from AQP1-null mice showed unaltered adhesion and proliferation; however, cell 13
ACCEPTED MANUSCRIPT migration was largely impaired and in vitro vessel formation was abnormal (Saadoun et al., 2005). Inversely, overexpression of AQP1 in transfected tumor cells increased cell migration
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2-3 fold (Hu & Verkman, 2006). The molecular role of AQP water permeability in cell migration is thought to reside in facilitating the formation of membrane protrusions at the
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leading edge (Papadopoulos et al., 2008). As a driving force, a local increase in the osmolarity by actin depolymerisation and ion transport initiates water influx (Condeelis, 1993; Lauffenburger & Horwitz, 1996; Schwab, 2001; Verkman, 2005). Inhibitors of AQP water
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permeability, thus, may be useful in anti-tumor therapy by interfering with tumor
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angiogenesis and tumor cell spreading. An apparently promising attempt was made in a nude mouse colon cancer model, which was treated with acetazolamide that was found to reduce
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AQP1 expression (Bin & Shi-Peng, 2011).
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High AQP5 expression levels have been associated with a worsened prognosis in non-small cell lung cancer (Chae et al., 2008). In vitro assays indicated that elevated AQP5 expression
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enhanced cell proliferation, migration, and invasion (Zhang et al., 2010). AQP5 was also
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found to promote proliferation and migration of human gastric carcinoma cells (Huang et al., 2013). Hence, AQP5 has been suggested as a prognostic cancer marker (Jung et al., 2011). AQP5 inhibitors might also be useful as anticancer therapeutics.
AQP3 is the major aquaglyceroporin in barrier tissues, such as the skin, (Matsuzaki et al., 1999) and lack of AQP3 affects hydration, elasticity, wound healing, and barrier function (Hara & Verkman, 2003). Elevated AQP3 expression levels are found in squamous cell skin carcinomas, whereas AQP3-null mice exhibit slower tumor growth and spreading (HaraChikuma & Verkman, 2008a, 2008b). The lack of glycerol permeability via AQP3 leads to a reduced cellular glycerol content and ATP biosynthesis, resulting in slow cell proliferation. Accordingly, therapeutic AQP3 inhibitors might interfere with skin tumor formation and 14
ACCEPTED MANUSCRIPT growth. Currently, cosmetics are marketed that contain inducers of AQP3 expression in keratinocytes in order to improve skin hydration. It is unclear at the moment whether and, if
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so, by what extent an increase in the carcinogenic risk is given when such cosmetics are used (Verkman, 2008). AQP3 was further shown to facilitate growth of human esophageal and oral
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squamous cell carcinoma (Kusayama et al., 2011). In this context, downregulation of AQP3 by siRNA increased the therapeutic effect of cisplatin and led to higher sensitivity of prostate
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cancer cells to experimental cryotherapy (Ismail et al., 2009).
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2.6 Immune system, hematocytes, parasite infections
Motility and rapid changes in cell volume are hallmarks of activated cells of the immune system. Dendritic cells are highly motile during antigen presentation and express AQP3 and
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AQP7 (Moon et al., 2004). AQP3 null mice exhibit developmental shifts and reduced motility
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in the dendritic cell subpopulations (Song et al., 2011). B and T lymphocytes initiate expression of AQP1, AQP3, and AQP5 upon activation, whereas AQP3 is already present in
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immature dendritic cells (Moon et al., 2004). In macrophages that have been activated by
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inflammation signals, deletion or inhibition of AQP1 results in reduced secretion of interleukin 1 (Rabolli et al., 2014). Similar to the work on AQPs in tumor biology, investigation of the role of AQPs in the immune response is just taking up speed. Since AQPs seem to fulfill roles mainly in activated and migrating immune cells it seems fair to propose that research on AQP inhibitors may lead to new approaches in anti-inflammatory therapy.
AQP9 function is linked to the treatment of a certain form of leukemia, i.e. acute promyelocytic leukemia; here, it is not water transport that is relevant but provision of an entry pathway for the drug arsenic trioxide, As2O3. When dissolved in water the compound forms As(OH)3 resembling in shape the glycerol molecule, which is a permeant of AQP9. Once inside the cytosol, arsenic drives the malignant promyelocytes into apoptosis (Dong, 15
ACCEPTED MANUSCRIPT 2002). The treatment possibilities by arsenic of other types of cancer including solid tumors are under investigation. In this regard, a lung adenocarcinoma cell line was identified that was
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resistant to arsenic trioxide due to downregulation of AQP9 expression (Lee et al., 2006).
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Pentavalent organic metalloids containing arsenic or antimony also are still the first line treatment of infectious diseases caused by Leishmania parasites, i.e. leishmaniasis (Mukhopadhyay et al., 2011). The compounds will be reduced to trivalent hydroxides by the
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human host cell and taken up by the parasite via an aquaglyceroporin, LmAQP1 in the case of
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Leishmania major (Gourbal et al., 2004; Mukhopadhyay et al., 2011). Resistant Leishmania strains have been isolated and are characterized by reduced AQP expression levels. Similarly, an aquaglyceroporin of Trypanosoma brucei parasites, TbAQP2 (Uzcategui et al., 2004),
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causing trypanosomiasis is required for drug uptake of pentamidine and melarsoprol (Alsford
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et al., 2012; Baker et al., 2012). It remains to be clarified how exactly the large and in the case
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transported.
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of pentamidine positively charged compounds make use of TbAQP2 in order to be
Aquaglyceroporins generally appear to predominate over water-specific AQPs in humanpathogenic parasites causing Chagas´ disease (Trypanosoma cruzi; Montalvetti et al., 2004), toxoplasmosis (Toxoplasma gondii; Pavlovic-Djuranovic et al., 2003), and malaria (Plasmodium spp.; Hansen et al., 2002). Their cellular functions are thought to reside in the compensation of osmotic stress, e.g. during kidney passages and transmission, in the release of waste metabolites, such as ammonia (Zeuthen et al., 2006) and methylglyoxal (PavlovicDjuranovic et al., 2006), and in the uptake of glycerol as a metabolic precurser for glycerolipid biosynthesis (Beitz et al., 2004; Song et al., 2012). Accordingly, they hold a good potential as novel drug targets.
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ACCEPTED MANUSCRIPT 2.7 Fat metabolism The human aquaglyceroporins AQP7 and AQP9 are expressed in adipose tissue and the liver,
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respectively (Hara-Chikuma et al., 2005; Kuriyama et al., 2002). AQP7 facilitates glycerol efflux from adipocytes during lipolysis (Maeda et al., 2004). As a consequence, AQP7 null
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mice become obese and insulin resistant due to functioning lipid accumulation but dysfunctional lipid degradation and release (Hibuse et al., 2005). In humans, mutations of AQP7 are associated with an inability to elevate plasma glycerol levels during exercise
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(Kondo et al., 2002). Increasing the glycerol permeability of adipocytes by acting on AQP7 or
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by other means might provide a novel access to fat reduction in obesity (Wang et al., 2006).
AQP9 can be considered as the liver counterpart of AQP7 in that it is responsible for the
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uptake of glycerol from the plasma, which is used for gluconeogenesis (Jelen et al., 2011;
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Jelen et al., 2012). In AQP9 null mice, plasma levels of glycerol and glycerolipids are increased and liver glucose biosynthesis is independent from plasma glycerol levels (Rojek et
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al., 2007; Jelen et al., 2011). Inhibition of AQP-facilitated glycerol uptake by the liver may be
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beneficial in the prevention of steatosis and consequences, such as steatohepatitis and cirrhosis (Calamita et al., 2012; Rodríguez et al., 2014).
The selection of examples described above show that, from a (patho-)physiological perspective, AQPs are promising drug targets. In order to test small molecule inhibitors permeability assay systems are required that are reliable, easy to handle, and open to upscaling processes in terms of increased throughput for screening larger compound libraries.
3. Assay systems for AQP function and inhibition
17
ACCEPTED MANUSCRIPT Determination of water or solute permeability is demanding and requires formation of two compartments separated by a membrane carrying the channel or transport protein of interest
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(Fig. 2). Compartmentation can be achieved by employing living cells in assay media or by using artificial systems, such as proteoliposome suspensions or black lipid membranes
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separating two buffer reservoirs. The establishment of an osmotic or chemical gradient initiates AQP-facilitated water or solute flux, respectively, across the membrane. Phenotypic effects, e.g. cell-growth, or biophysical measures, such as light scattering, fluorescence,
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radiotracer decay, or surface plasmon resonance, serve as a readout. AQP inhibitors can be
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found by adding test compounds prior to the assay. The challenge lies in the construction of a
3.1 Phenotypic AQP assays
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robust setup that is ready for automation and high-throughput screenings.
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To expose an AQP-dependent growth phenotype, yeast cells can be challenged by osmotic stress, forced to take up toxic compounds, or provided access to nutrients (Fig. 2A). Yeast
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strains are rendered osmo-sensitive and cease to grow under hypertonic conditions when an
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aquaglyceroporin is expressed and endogenous glycerol acting as osmotic pressure regulating compatible solute cannot accumulate (Pettersson et al., 2006). Yeast growth will also be affected if the AQP facilitates uptake of toxic arsenite (Liu et al., 2002; Wu et al., 2010), methylamine (Beitz et al., 2006; Zeuthen et al., 2006), or hydrogen peroxide (Bienert et al., 2007; Almasalmeh et al., 2014). A positive growth selection of yeast is possible by heterologous expression of an ammonia-facilitating AQP in a strain that lacks endogenous ammonium transporters (Holm et al., 2005). Phenotypic growth assays can be carried out in the presence or absence of putative AQP inhibitors either on solid agar media with photographic imaging of the cell density (Wu et al., 2008) or in liquid cultures by documenting the turbidity over time (Almasalmeh et al., 2014). The latter yields more quantitative data when integrating the areas under the curves from which IC50 values can be 18
ACCEPTED MANUSCRIPT estimated. A limiting factor of phenotypic yeast growth assays is the incubation time of several days during which added compounds are likely to be lost due to chemical instability
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or metabolism by yeast enzymes. Further, each compound has to be analyzed with respect to
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cytotoxicity in order to recognize false positives.
In this sense, biophysical assays appear more suitable for compound screening because measurements are carried out on a much shorter, seconds to minutes timescale. However, they
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require sophisticated instrumentation and sample handling, which hamper automation and
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throughput.
3.2 Biophysical AQP assays
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The first cell-based biophysical assays for water permeability of specific AQPs were carried
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out using Xenopus laevis oocytes, which were injected with cRNAs encoding the AQP of interest (Preston et al., 1992). Within four days maximal protein levels are obtained and the
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oocytes are challenged by a hypotonic shock or by isotonic replacement of salt from the
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buffer by a solute to test for instance for glycerol permeability. Oocyte swelling due to water influx will be video-monitored and the rate is calculated from the increase of the area covered by the oocyte (Fig. 2B). The large size of the spherical oocytes of approximately 1.2 mm in diameter yields relatively low swelling rates requiring monitoring times of around one minute (Beitz et al., 2009). Parallelization is thinkable by placing the oocytes in 96-well plates and imaging the whole set-up. However, the preparation procedure of the Xenopus oocytes, i.e. operation on a female frog, selection of mature stages, proteolysis of the surrounding vitelline layer, and individual cRNA injection limits higher throughput screening.
The transition to easier to obtain, yet smaller cells, such as cultured epithelial cells, with a width in the range of 10-20 µm and a flat shape, or to erythrocytes, yeast cells, or 19
ACCEPTED MANUSCRIPT proteoliposomes of a few hundred nanometers in diameter calls for more rapid (seconds and
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subseconds range) and precise determination of volume changes.
Suspensions of cells or proteoliposomes can be analyzed using a stopped-flow apparatus by
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recording the particle size-dependent intensity of scattered light (Fig. 2B). The cells or proteoliposomes are rapidly mixed with an osmolyte or solute containing buffer and data acquisition is run for several seconds. Volume increase results in a decrease of the light
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scattering intensity whereas shrinkage leads to elevated signals. Human erythrocytes are well
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suited for this assay because they form non-adherent cell suspensions and contain high levels of water-specific AQP1 and the aquaglyceroporin AQP3 in their native membrane environment facilitating direct evaluation of respective modulators (Martins et al., 2012). The
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number of AQP1 monomers in human erythrocytes was estimated via quantitative
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immunoblotting to 200,000 monomers per erythrocyte (Denker et al., 1988). This lies in the same range as the number of water channels estimated by biophysical analysis of 270,000
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(Solomon et al., 1983). Heterologously expressed AQPs in baker’s yeast, Saccharomyces
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cerevisiae, are a reasonable alternative. Enzymatic zymolyase digestion of the rigid yeast cell wall prior to the assay provides the required elasticity of the obtained protoplasts for the assay (Bertl et al., 1998). Generally, the stopped-flow light scattering technique provides the necessary accuracy and reproducibility to determine IC50 or Ki values. However, the devices typically hold a single measuring chamber and are, thus, better suited for careful evaluation of candidate inhibitors than for running large screening programs (Levin et al., 2007).
Most native human cells are adherent and examination of volume changes is possible by microscopy (Fig. 2B). Since the trace length of a beam of light through a cell layer is a onedimensional indicator of the cell volume, interferometric microscopy can be used to follow volume changes of AQP expressing cells (Farinas & Verkman, 1996). Alternatively, 20
ACCEPTED MANUSCRIPT recordings of volume-dependent fluorescence intensity of entrapped fluorophores can be used to monitor cell swelling and shrinking (Farinas et al., 1995; Soveral et al., 2007; Mola et al.,
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2009; Madeira et al., 2010). In this sense, the chloride-sensitive yellow fluorescent protein, YFP-H148Q/V163S indicates cell swelling via dilution of cytoplasmic chloride
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concentrations and decreasing fluorescence intensity (Galietta et al., 2001; Baumgart et al., 2012; Esteva-Font et al., 2013).
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If epithelial cells form a tight layer, transcellular water flux can be quantified using an Ussing
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chamber (Fig. 2B). Loss and gain of volume on the apical and the basolateral sides of the cell membrane as a result of a transepithelial osmotic gradient can be measured as changes in the electrical conductivity and converted into a fluid transport rate (µl cm–2 h–1) (Edelman &
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Miller, 1991). A more artificial, yet, pure lipid-protein setup, i.e. black lipid membranes,
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involves a single lipid bilayer facing a buffer chamber on either side (Fig. 2B). Ag/AgCl electrodes register electrolyte dilutions close to the bilayer due to water flux via reconstituted
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AQPs as changes in the electrochemical potentials (Bárány-Wallje et al., 2005). The solute
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permeability of the aquaglyceroporin of malaria parasites has been analyzed using surfacetethered proteoliposomes. The process of solute loading of the proteoliposomes led to changes is the refractive index and was measured by surface plasmon resonance (Brändén et al., 2010).
3.3 High-throughput AQP assays In order to identify hits from a high-throughput inhibitor screening a definitive yes/no statement from single time point measurements is desired. One successful approach for identifying inhibitors of the urea transporter type B, UT-B, employed native, AQP1containing erythrocytes in an assay with cell lysis as a readout (Levin et al., 2007). The erythrocytes were preloaded in hypertonic acetamide solution and subsequently exposed to an 21
ACCEPTED MANUSCRIPT isotonic buffer without the compound resulting in acetamide efflux due to the chemical outward gradient. In the presence of a UT-B inhibitor, the entrapped acetamide maintained
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the increased intracellular osmolarity and led to water influx via AQP1 and eventually cell lysis. This way, a library of about 50,000 small molecules was screened. Inhibitors of AQP1
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should be identifiable in a similar way by monitoring the resistance towards erythrocyte lysis in hypotonic buffers (Table 1).
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A variation of the erythrocyte lysis assay was recently applied to yeast cells. Here the AQP-
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dependent freeze tolerance of the cells was used as readout (Ahmadpour et al., 2014; To et al., 2015). Yeast cells expressing a water-permeable AQP recover better from shock freezing and thawing, which can be monitored within hours by a spectroscopic viability assay. AQP
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inhibitors would be identified by reduced viability. The yeast system reproduced the findings
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of an AQP1 inhibitor screen based on microscopy of human cells (Mola et al., 2009) and
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and costs.
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appears quite attractive with respect to free selection of the AQP to be expressed, robustness,
4. Towards therapeutic modulation of AQP function The ability of small molecules to cause a pharmacological effect is based on the interaction with their target proteins. The physicochemical properties of both, small molecule and target, determine whether the interaction is of sufficient specificity and affinity in order to generate the desired efficacy and to reduce the risk of unwanted side effects. Interaction sites of small molecules are based on the type and spatial orientation of their functional groups and usually form hydrogen bonds, and ionic, hydrophobic, and cation- interactions; covalent suicideinhibitors are rare. The affinity rises with the number of interactions, the rigidity of the small molecule, and with the degree of the shielding from the aqueous solvent because water molecules will compete for the interaction sites within a protein. Hence, a deep binding 22
ACCEPTED MANUSCRIPT pocket that is optimally filled by a drug substance produces the best affinity. It further turned out that it is favorable if the compound provides no more than 5 hydrogen bond donor and 10
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hydrogen bond acceptor sites and if the molecular mass is less than 500 Da (rule of five;
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Lipinski et al., 2001).
In order to become useful as a drug substance, the small molecule has to comply with certain pharmacokinetic requirements. First of all, it is necessary that the compound reaches the
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target. While only a few compounds can enter circulation via transport proteins, the majority
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of compounds will have to exhibit sufficient lipophilicity to enable transmembrane diffusion. A measure for the lipophilicity of drug substances is the partition-coefficient, P, representing the equilibrium concentration ratio of the compound in 1-octanol and water. The diffusion of
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small molecules through the tissue is proportional to the logarithm of the partition-coefficient
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described by logP. Drug substances with a good oral bioavailability and good distribution
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behavior usually exhibit a logP smaller or close to 5 (Lipinski et al., 2001).
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4.1 AQP protein structures as targets for small molecules Since the AQP protein family is ancient and shares a highly conserved fold especially in the central parts of the conducting channel, finding a specific small molecule inhibitor could be demanding. AQPs form homotetramers with four individual water/solute conducting channels in the protomers. Two filter regions are located within the channel interior, i.e. the aromatic/arginine (ar/R) selectivity filter towards the extracellular pore entry and the central Asn-Pro-Ala (NPA) region (Fig. 3, top). The ar/R region represents the narrowest constriction and selects permeants by size (Beitz et al., 2006). Together with the NPA region inorganic cations and protons are perfectly prevented from leaking through the AQP (Wu et al., 2009; Wree et al., 2011). The inflexible and tight, funnel-shaped inner structure leaves only little space for potential inhibitors (Fig. 3A). At the same time, it is necessary that an inhibitor 23
ACCEPTED MANUSCRIPT binds with high affinity to displace the excessively abundant water molecules. Affinity is increased if interactions with the protein are shielded against water by lipophilic moieties of
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the inhibitor. However, in this case it is likely that the compound would be oversized to fit the AQP channel. Generally, aquaglyceroporins appear to have deeper and wider vestibules than
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water-specific AQPs (Fig. 3, bottom).
An accompanying table to the text below lists candidate compounds, concentration ranges,
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test systems, and outcome with respect to AQP inhibition (Table 1).
4.2 AQP inhibition by transition metals
Long before the discovery of the AQPs, it was found that mercuric chloride and
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organomercurials inhibit water and solute permeability of native erythrocytes (Fig. 4; Macey
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& Farmer, 1970). In fact, this notion hinted at the proteinaceous nature of the elusive water channel and further boosted the search for the AQPs. Later, direct inhibition of the isolated
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erythrocyte water channel by mercuric chloride was shown in Xenopus oocytes expressing
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human AQP1 (Preston et al., 1992) and the mercurial-sensitive site was located to Cys189 at the extracellular pore constriction (Preston et al., 1992; Preston et al., 1993; Zhang et al., 1993; Ozu et al., 2011). AQP2 is equally sensitive to mercurials and also carries a cysteine at the respective position (Fushimi et al., 1993), whereas AQP4 with an alanine at the critical site was identified as a first mercurial-insensitive water channel (Hasegawa et al., 1994). AQP4, when reconstituted in proteoliposomes, however, exhibited mercurial-sensitivity suggesting participation of a cysteine at the intracellular surface (Yukutake et al., 2008). Other metal-based compounds containing silver, gold, copper, nickel, lead, or tin were found to inhibit aquaporins as well (Niemietz & Tyerman, 2002; Zelenina et al., 2003; Zelenina et al., 2004; Yang et al., 2006; Mola et al., 2009). The coordination gold(III) complexes auphen (Fig. 4), audien, aubipy, and auterpy selectively inhibit glycerol and water transport in red 24
ACCEPTED MANUSCRIPT blood cells via the aquaglyceroporin AQP3 (without affecting AQP1) at low micromolar concentrations (Martins et al., 2012; Martins et al., 2013). Docking models as well as
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mutagenesis identified Cys40 located towards the extracellular face of AQP3 to coordinate the gold complexes, whereas Cys189 in AQP1 (Fig. 3A) appeared inaccessible for these
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larger compounds (Martins et al., 2012; Martins et al., 2013; Serna et al., 2014). The same gold(III) complexes where found to inhibit also AQP7 in adipocytes (Madeira et al., 2014), however in this case other binding modes for gold(III) should be in place since this isoform
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lacks Cys40. Thus, binding to Cys residues is not always the primary mechanism of inhibition
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for metal-based compounds, but others may be in place and further investigation is necessary.
Metallo-compounds are the most effective AQP inhibitors available. This is certainly due to
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the high affinity of a metal-thiolate complex which is partially covalent yielding binding
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energies well above 100 kcal mol–1, i.e. approximately one third that of a fully covalent bond (Solomon et al., 2006). The affinity problem in AQP inhibition seems, thus, solved. Using
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such potent binders, however, one has to make sure that high selectivity is given in order to
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prevent quasi-permanent modification of uninvolved proteins and the risk of side effects. Hence, effects of simple inorganic mercuric chloride and salts of the other mentioned metals are a topic in toxicology but certainly not in pharmacology. A larger scaffold in the organic part of the metal-complex, as it is given in the auphen-like compounds, may provide options for molecule modifications that lead to the required selectivity (Martins et al., 2012). There are established examples of heavy metal-based compounds, such as auranofin in the treatment of rheumatoid arthritis, or the anticancer drug cisplatin showing that, in principle, metallodrugs are of therapeutic value (Mjos & Orvig, 2014).
4.3 AQP inhibition by small organic molecules
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ACCEPTED MANUSCRIPT The outcome of studies on non metal-based small molecule inhibitors targeting AQPs is mixed and apparently depends on the assay system. One of the first compounds for which
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AQP inhibition was published is the quaternary nitrogen compound tetraethylammonium (TEA; Brooks et al., 2000; Detmers et al., 2006). Experiments with AQP-expressing Xenopus
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oocytes yielded significant, dose dependent and reversible inhibition of water permeability of AQP1, AQP2, and AQP4 (Brooks et al., 2000; Detmers et al., 2006). Additionally, the tetraethylammonium analogue tetrapropylammonium (TPrA) was effective on AQP1
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suggesting that quaternary nitrogen compounds represent a class of AQP blockers. Mutational
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studies identified Tyr186 of the extracellular loop E of AQP1 to be required for tetraethylammonium-inhibition (Brooks et al., 2000; Detmers et al., 2006; Müller et al., 2008). At 4 µM, tetraethylammonium inhibited AQP1 water permeability of Xenopus oocytes
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by 46%. Inhibition was also seen in AQP1-expressing Madin-Darby canine kidney cells
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(MDCK; Yool et al., 2002). However, when erythrocytes carrying native AQP1 were tested, tetraethylammonium and tetrapropylammonium showed no significant inhibition even at 10
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mM concentration (Yang et al., 2006). Similarly, stably AQP1-expressing Fisher rat thyroid
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(FRT) epithelial cells were not affected by 1 mM tetraethylammonium (Yang et al., 2006).
Established drug compounds mainly from the various classes of diuretics were tested for AQP inhibition. The diuretic carbonic anhydrase inhibitor acetazolamide that is now used in the treatment of glaucoma and altitude sickness showed significant, dose-dependent, low micromolar inhibition of AQP1 in Xenopus oocytes and in transfected human embryonic kidney 293 cells (HEK293; (Ma et al., 2004; Gao et al., 2006; Seeliger et al., 2013). Again, the inhibiting effect was absent when using erythrocytes and stably AQP1 expressing FRT epithelial cells (Yang et al., 2006). Acetazolamide was further ineffective on purified and reconstituted AQP1 in proteoliposomes; yet, at 1.25 mM reversible, 50% inhibition of reconstituted AQP4 was seen (Tanimura et al., 2009) supporting earlier observations of AQP4 26
ACCEPTED MANUSCRIPT inhibition in Xenopus oocytes (Huber et al., 2007). However, when using transfected FRT cells or glial cells that natively express AQP4, the acetazolamide effect could not be
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reproduced (Yang et al., 2008). At the molecular level this effect may be explained considering that a direct and functional interaction between AQP1 and carbonic anhydrase has
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been recently described (Vilas et al. 2015).
Another approach was to replace the cytosol of Xenopus oocytes with a defined buffer
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(emptied-out oocytes) and application of the loop diuretic furosemide to the intracellular face
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of AQP1. This treatment led to inhibition of AQP1, whereas extracellular furosemide did not affect water permeability (Ozu et al., 2011). A second sulfamyl loop diuretic, bumetanide, significantly reduced the osmotic water flux via AQP4 in Xenopus oocytes and the efficacy
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was increased when the compound was injected (Migliati et al., 2009). A bumetanide
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derivative, AqB013, was even more effective and inhibited AQP1 and AQP4 with an IC50 of
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20 µM when it was applied extracellularly (Migliati et al., 2009).
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Several antiepileptic drugs, topiramate, phenytoin, lamotrigine, as well as triptans, sumatriptan, rizatriptan, were reported to act as inhibitors of AQP4 in the Xenopus oocyte system with double-digit micromolar IC50 (Huber et al., 2009a; Huber et al., 2009b). However, when tested at 100 µM on erythrocytes and stably AQP4-transfected FRT cells no inhibition was observed (Yang et al., 2008). A virtual screening approach using compounds of the ZINC database (Seeliger et al., 2013) yielded several potential extracellular AQP1 inhibitors of which three structurally unrelated compounds inhibited osmotic swelling of AQP1-expressing Xenopus oocytes at low micromolar concentrations. In the same study, the compounds were ineffective in blocking AQP1-mediated water permeability of human erythrocytes.
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ACCEPTED MANUSCRIPT A likely explanation for the variation in efficacy in different assay system is the number of functional AQP channel proteins present in the respective plasma membrane. Cells exhibiting
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low expression levels should be more sensitive to compounds that bind with low affinity. Care should be taken, that the assay conditions mirror the physiological situation not only
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with respect to the selected AQP isoform but regarding its abundance in the cell membrane, which can be very high (Denker et al., 1988).
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An automated screening assay based on fluorescence changes in calcein-loaded fibroblasts
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and astrocytes that natively express AQP1 and AQP4, respectively, yielded several putative AQP inhibitors (Mola et al., 2009). Re-evaluation within the same study of four hits, NSC164914 and NSC168597 (metallo-compounds; Fig. 4), NSC301460 and NSC670229
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(metal-free; Fig. 4), using erythrocytes and membrane vesicles from transfected AQP4-
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expressing cells confirmed EC50 values in the low micromolar range. While NSC301460 (trychopolyn B) is a fairly complex peptide with a molecular mass of 1188 Da, NSC670229 is
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more drug-like. Recently, the results on AQP1 inhibition were independently reproduced
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using a freeze-thaw assay using yeast cells (To et al., 2015). This latter study produced two additional compounds that inhibited AQP1 water-permeability of human erythrocytes.
With respect to aquaglyceroporins, a small screening approach using a fluorescencequenching assay identified compounds that efficiently inhibited AQP9-mediated glycerol permeability in primary hepatocyte cultures. The IC50 values of these substances were between 0.15 and 4.5 µM (Jelen et al., 2011). Based on these data, putative binding sites were evaluated by point mutations of AQP9 and several more compounds were found to be effective with single-digit micromolar IC50 values in AQP9-expressing Chinese hamster ovary cells (Wacker et al., 2013).
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ACCEPTED MANUSCRIPT 4.4 Inhibition of the interaction of AQP4 with autoantibodies AQP4 provides extracellular epitopes that are targeted by autoantibodies in the degenerative
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disease neuromyelitis optica (NMO). The aim here is not to modulate AQP4 water permeability but to prevent binding of NMO-IgG antibodies to AQP4. A monoclonal
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antibody, aquaporumab, was generated against extracellular domains of AQP4, which does not affect water permeability but blocks the pathological interaction with NMO-IgG due to steric hindrance as tested in cultured cells, spinal cord slices, and in vivo mouse models of
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NMO (Tradtrantip et al., 2012a). Further, a high-throughput screening was set up to identify
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small molecules that block binding of NMO-IgG to human AQP4. In the assay, recombinant monoclonal NMO-IgG was added to transfected FRT cells that stably express AQP4 in the presence of test compounds (Tradtrantip et al., 2012b). Out of 60,000 compounds the antiviral
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arbidol and the flavonoid tamarixetin were picked up that blocked NMO-IgG binding to
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AQP4 with 5 μM IC50. Statements on the safety and efficacy of both, aquaporumab and
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possibly small NMO-IgG interacting molecules, need to await clinical trials.
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4.5 AQP gene replacement therapy A complication connected to radiation therapy in head and neck cancer is that salivary tissue is destroyed in the process and respective patients suffer severely from dry a mouth leading to ulcers, inflammation, infections, tooth decay, and swallowing difficulties (Baum et al., 2012; Lee et al., 2015). Since the atrophy of laryngeal cells is not treatable in a classical way, gene technological attempts have been made in rat irradiation models to bring AQP1 to expression and restore saliva production (Baum et al., 2012). In a recent human phase I clinical trial, AQP1 cDNA was transferred to eleven individuals by an adenovirus. The study procedures were well tolerated over 42 days. Nevertheless, the risks connected to the use of an adenoviral vector impeded an advancement to phase II trials (Wang et al., 2015). The recent successful
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ACCEPTED MANUSCRIPT ultrasound-assisted, nonviral gene transfer of AQP1 to an irradiated swine model may open
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up new options for human trials.
5. Conclusions
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The great potential of AQPs to serve as drug targets has been recognized early after their discovery (Beitz & Schultz, 1999). The development of small molecules that specifically modulate AQP function is challenged by spatial restrictions in the protein structure and by the
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high protein abundance in the plasma membranes. Assay systems are demanding with respect
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to instrumentation and handling. The assay outcome depends on the AQP expression level with native cells, such as erythrocytes, being harder to affect. A first set of inhibitors appears to reproducibly inhibit AQPs in independent assay systems indicating that small molecule
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inhibition of AQPs is possible. An alternative approach would be not to target the AQP
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protein directly but to act on intracellular trafficking or signal transduction pathways, such as
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in the case of the AQP2-modulating vaptan class of vasopressin receptor antagonists.
Conflict of Interest Statement The authors declare that there are no conflicts of interest.
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ACCEPTED MANUSCRIPT Figure legends Figure 1 Physiological situations that require AQP water or glycerol facilitation across cell
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membranes.
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Figure 2 Systems for assaying AQP function. A. Phenotypic assays monitor cell growth or cell death during provision of AQP passing nutrients or toxins, or by exposing the cells to osmotic stress on an hours to days time scale. B. Biophysical assays detect volume changes
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upon osmotic challenges within seconds or subseconds using various physical measures as a
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readout.
Figure 3 Crystal structures of pharmacologically relevant AQPs. AQP1 (PDB# 1FX8), AQP4
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(2D57), and AQP5 (3D9S) are water-specific, PfAQP (3C02) from the malaria parasite
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Plasmodium falciparum is an aquaglyceroporin. The water (blue shading) and glycerol (orange) permeation pathways and vestibules are colored to indicate sites where inhibitor
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binding would directly interfere with AQP function. Diameters of the main constriction site,
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i.e. the aromatic/arginine (ar/R) selectivity filter, are indicated as well as the minimal and maximal width and area of a section through the extracellular vestibule 8 Å above the ar/R region.
Figure 4 Chemical structures of AQP inhibitors. Shown are molecules which were confirmed to be effective by independent investigators using different assay systems. pCMBS – pchloromercuribenzylsulfonate, NSC – numbering scheme according to the Cancer Chemotherapy National Service Center, USA.
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Wu, B., Steinbronn, C., Alsterfjord, M., Zeuthen, T. & Beitz, E. (2009). Concerted action of two cation filters in the aquaporin water channel. EMBO J 28, 2188–2194. Wu, B., Song, J. & Beitz, E. (2010). Novel channel enzyme fusion proteins confer arsenate resistance. J Biol Chem 285, 40081–40087. Yang, B., Gillespie, A., Carlson, E.J., Epstein, C.J. & Verkman, A.S. (2001). Neonatal mortality in an aquaporin-2 knock-in mouse model of recessive nephrogenic diabetes insipidus. J Biol Chem 276, 2775–2779. Yang, B., Kim, J.K. & Verkman, A.S. (2006). Comparative efficacy of HgCl2 with candidate aquaporin-1 inhibitors DMSO, gold, TEA+ and acetazolamide. FEBS Lett 580, 6679–6684. Yang, B., Zhang, H. & Verkman, A.S. (2008). Lack of aquaporin-4 water transport inhibition by antiepileptics and arylsulfonamides. Bioorg Med Chem 16, 7489–7493. Yang, B., Zhao, D. & Verkman, A.S. (2009). Hsp90 inhibitor partially corrects nephrogenic diabetes insipidus in a conditional knock-in mouse model of aquaporin-2 mutation. FASEB J 23, 503–512. Yasui, M., Hazama, A., Kwon, T.H., Nielsen, S., Guggino, W.B. & Agre, P. (1999). Rapid gating and anion permeability of an intracellular aquaporin. Nature 402, 184–187.
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ACCEPTED MANUSCRIPT Yool, A.J., Brokl, O.H., Pannabecker, T.L., Dantzler, W.H. & Stamer, W.D. (2002). Tetraethylammonium block of water flux in Aquaporin-1 channels expressed in kidney thin limbs of Henle's loop and a kidney-derived cell line. BMC Physiol 2, 4.
PT
Yukutake, Y., Tsuji, S., Hirano, Y., Adachi, T., Takahashi, T., Fujihara, K. et al. (2008). Mercury chloride decreases the water permeability of aquaporin-4-reconstituted proteoliposomes. Biol Cell 100, 355–363.
SC RI
Zelenina, M., Bondar, A.A., Zelenin, S. & Aperia, A. (2003). Nickel and extracellular acidification inhibit the water permeability of human aquaporin-3 in lung epithelial cells. J Biol Chem 278, 30037–30043. Zelenina, M., Tritto, S., Bondar, A.A., Zelenin, S. & Aperia, A. (2004). Copper inhibits the water and glycerol permeability of aquaporin-3. J Biol Chem 279, 51939–51943.
NU
Zeuthen, T., Wu, B., Pavlovic-Djuranovic, S., Holm, L.M., Uzcategui, N.L., Duszenko, M. et al. (2006). Ammonia permeability of the aquaglyceroporins from Plasmodium falciparum, Toxoplasma gondii and Trypansoma brucei. Mol Microbiol 61, 1598–1608.
MA
Zhang, D., Vetrivel, L. & Verkman, A.S. (2002). Aquaporin deletion in mice reduces intraocular pressure and aqueous fluid production. J Gen Physiol 119, 561–569. Zhang, H. & Verkman, A.S. (2010). Aquaporin-1 tunes pain perception by interaction with Na(v)1.8 Na+ channels in dorsal root ganglion neurons. J Biol Chem 285, 5896–5906.
ED
Zhang, R., van Hoek, A.N., Biwersi, J. & Verkman, A.S. (1993). A point mutation at cysteine 189 blocks the water permeability of rat kidney water channel CHIP28k. Biochemistry 32, 2938–2941.
AC
CE
PT
Zhang, Z., Chen, Z., Song, Y., Zhang, P., Hu, J. & Bai, C. (2010). Expression of aquaporin 5 increases proliferation and metastasis potential of lung cancer. J Pathol 221, 210–220.
43
MA
NU
SC RI
PT
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
Figure 1
44
ED
MA
NU
SC RI
PT
ACCEPTED MANUSCRIPT
AC
CE
PT
Figure 2
45
MA
NU
SC RI
PT
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
Figure 3
46
SC RI
PT
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
MA
NU
Figure 4
47
ACCEPTED MANUSCRIPT Table 1. incubation time (min)
extent of inhibitio n
300 and 3000
5
significa nt
300
0 (only in assay solution) 5
significa nt
300
NU
30 2.5
significa nt 68 %
tetraethylammoni um
acetazolamide (carboanhydrase inhibitor)
2.5
75 %
10
not significa nt
PT
AC
HAuCl3
CE
100
100
3.9
6
ED
15
silver sulfadiazine
referenc e
Xenopus oocytes
Preston et al., 1992 Zhang et al., 1993
Xenopus oocytes
10
MA
(50 mg/ml)
test system
Xenopus oocytes
significa nt
15 pCMB-conjugated dextran AgNO3
IC50 (μM )
PT
AQP1 HgCl2
test conc. (μM)
SC RI
test compound
15
1.24
significa nt
15
46 %
1000
240
34.2 %
10000
240
not significa nt
1000
15
10
15
1.4
81 %
15
5.5
48
mouse and human erythrocytes human erythrocytes human erythrocytes
14
15
mouse and human erythrocytes human erythrocytes human erythrocytes
mouse and human erythrocytes Xenopus oocytes Xenopus oocytes AQP1-MDCK cells mouse and human erythrocytes AQP1-FRT cells Xenopus oocytes Xenopus oocytes
Preston et al., 1993 Yang et al., 2006 Zhang et al., 1993 Niemietz & Tyerman , 2002 Yang 2006 Niemietz & Tyerman , 2002 Martins et al., 2012 Yang et al., 2006 Brooks et al., 2000 Detmers et al., 2006 Yool et al., 2002 Yang et al., 2006
Ma et al., 2004 Seeliger et al., 2013
ACCEPTED MANUSCRIPT 30
39 %
AQP1-HEK293
2000
240
not significa nt
1000
15
1250
30
mouse and human erythrocytes AQP1-FRT cells AQP1proteoliposom es Xenopus oocytes human erythrocytes
15 10 – 20
compound 2
15 10 – 20
compound 3
15
5 and 60 60 – 120
significa nt
15
AC
NSC670229
Ozu et al., 2011
20
5
significa nt
Xenopus oocytes
15
40 %
significa nt 49 significa nt 28
20
tetraethylammoni um
emptied-out Xenopus oocytes Xenopus oocytes
27
15
300
Seeliger et al., 2013
significa nt
15
20
AQP2 HgCl2
Xenopus oocytes human erythrocytes
significa nt
15
NSC301460
17.5
rat erythrocytes yeast freezethaw assay rat erythrocytes yeast freezethaw assay rat erythrocytes yeast freezethaw assay rat erythrocytes yeast freezethaw assay
20
NSC168597
Seeliger et al., 2013
40
20
49
Tanimur a et al., 2009 Seeliger et al., 2013
Xenopus oocytes human erythrocytes
not significa nt ca. 66 %
PT
10 (intracellula r) 20
CE
NSC164914
10 – 20
Gao et al., 2006 Yang et al., 2006
17.0
not significa nt
ED
4
not significa nt
MA
20
AqB013
8.1
NU
20
furosemide (loop diuretic)
SC RI
compound 1
not significa nt
PT
100
6.2
Xenopus oocytes
Migliati et al., 2009 Mola et al., 2009 To et al., 2015 Mola et al., 2009 To et al., 2015 Mola et al., 2009 To et al., 2015 Mola et al., 2009 To et al., 2015 Fushimi et al., 1993 Detmers et al., 2006
ACCEPTED MANUSCRIPT
Aubipy
30
AubipyMe
30
AubipyNH2
30
Auterpy
30
Cuphen
30
AQP4 HgCl2
CE
1250
AC
acetazolamide (carboanhydrase inhibitor)
50 %
15
57 %
5
53.3 %
120
80 % at 20 µM not significant
PT
tetraethylammoni um
5
ED
5
human erythrocytes
16 .6
human erythrocytes
PT
30
0. 8
2. 3
human erythrocytes
SC RI
Audien
89 % (glycerol) at 100 µM 79 % (glycerol) at 50 µM 92,7 % (glycerol) at 10 µM 93,1 % (glycerol) at 10 µM 80 % (glycerol) at 10 µM 89 % (glycerol) at 10 µM 89 % (glycerol) at 1000 µM
NU
10
MA
AQP3 Auphen
1. 0
human erythrocytes
2. 9
human erythrocytes
1. 0
human erythrocytes
81 .9
human erythrocytes
9. 8
AQP4proteoliposom es Xenopus oocytes
0. 9
AQP4 proteoliposom es Xenopus oocytes AQP4-FRT cell membrane vesicles AQP4-FRT cell monolayers native glial cells Xenopus oocytes
100
15
5 (intracellula r)
120 – 240
significant
2-(nicotinamido)1,3,4-thiadiazole
120
67 % at 20 μM
3. 1
Xenopus oocytes
sumatriptan (H1-recept. agonist; anti-migraine)
120
54 % at 20 μM
11
Xenopus oocytes
15
not significant
furosemide (loop diuretic)
100
50
AQP4-FRT cell membrane vesicles AQP4-FRT cell
Martins et al., 2012 Martins et al., 2012 Martins et al., 2013 Martins et al., 2013 Martins et al., 2013 Martins et al., 2013 Martins et al., 2013 Yukutak e et al., 2008 Detmers et al., 2006 Tanimur a et al., 2009 Huber et al., 2007 Yang et al., 2008
Migliati et al., 2009 Huber et al., 2009a Huber et al., 2009a Yang et al., 2008
ACCEPTED MANUSCRIPT 52 % at 20 μM
20
120
68 %
100
15
not significant
20
120
23 %
120
67 % at 20 μM 75 % at 100 μM not significant
10
48 % at 20 μM 48 % at 100 μM not significant
58 % at 20 μM 60 % at 100 μM not significant
topiramate (antiepileptic drug)
15
ED 120
PT
zonisamide (antiepileptic drug)
15
AC
CE
100
phenytoin (antiepileptic drug)
oxcarbazepine (antiepileptic drug) lamotrigine (antiepileptic drug)
120
100
15
20
120
33 %
120
54 % at 20 μM 64 % 100 μM not significant
100
monolayers Xenopus oocytes
15
51
Huber et al., 2009a Huber et al., 2007 Yang et al., 2008
Yang et al., 2008
3. 3
AQP4-FRT cell membrane vesicles AQP4-FRT cell monolayers native glial cells Xenopus oocytes
Yang et al., 2008
9. 8
AQP4-FRT cell membrane vesicles AQP4-FRT cell monolayers Xenopus oocytes AQP4-FRT cell membrane vesicles AQP4-FRT cell monolayers Xenopus oocytes
Yang et al., 2008
PT
Xenopus oocytes AQP4-FRT cell membrane vesicles AQP4-FRT cell monolayers Xenopus oocytes Xenopus oocytes
MA
100
2. 9
SC RI
compound 4
120
NU
rizatriptan (H1-recept. agonist; anti-migraine) 6-ethoxybenzothiazole-2sulfonamide
8. 1
Xenopus oocytes AQP4-FRT cell membrane vesicles AQP4-FRT cell
Huber et al., 2007 Huber et al., 2009b
Huber et al., 2009b
Huber et al., 2009b
Huber et al., 2009b Huber et al., 2009b Yang et al., 2008
ACCEPTED MANUSCRIPT 120
40 %
carbamazepine10,11-epoxide (antiepileptic drug)
20
120
40 %
bumetanide (loop diuretic)
100 (extracellula r) 50 (intracellula r)
60 – 120 (extracellula r) 120 – 240 (intracellula r) 60 – 120
significant
NU
AqB013
MA
AQP9 RF03176 S14838
HTS13286
1.3 1.5
0.15 410
AC
CE
ID1 – ID6
20
3.0
PT
HTS13772
Xenopus oocytes
4.5
ED
CD05595
monolayers Xenopus oocytes
PT
20
52
Huber et al., 2009b Huber et al., 2009b
Xenopus oocytes
Migliati et al., 2009
Xenopus oocytes
Migliati et al., 2009
AQP9-CHO cells AQP9-CHO cells AQP9-CHO cells AQP9-CHO cells AQP9-CHO cells AQP9-CHO cells
Jelen et al., 2011 Jelen et al., 2011 Jelen et al., 2011 Jelen et al., 2011 Jelen et al., 2011 Wacker et al., 2013
SC RI
valproic acid (valproic acid)