Inhibition of Aquaporin 4 by antiepileptic drugs

Available online at www.sciencedirect.com

Bioorganic & Medicinal Chemistry 17 (2009) 418–424

Inhibition of Aquaporin 4 by antiepileptic drugs Vincent J. Huber,a Mika Tsujita,a Ingrid L. Kweeb and Tsutomu Nakadaa,b,* a

Center for Integrated Human Brain Science, Brain Research Institute, University of Niigata, 1 Asahimachi, Niigata 951-8585, Japan b Department of Neurology, University of California, Davis, CA 95616, USA Received 11 November 2007; revised 18 December 2007; accepted 19 December 2007 Available online 4 January 2008

Abstract—The potential of antiepileptic drugs (AEDs) to inhibit the water transport properties of Aquaporin 4 (AQP4) was investigated using a combination of in silico and in vitro screening methods. Virtual docking studies on 14 AEDs indicated a range of docking energies that spanned approximately 40 kcal/mol, where the most stabilized energies were consistent with that of the previously identified AQP4 inhibitor acetazolamide. Nine AEDs and one bio-active metabolite were further investigated in a functional assay using AQP4 expressing Xenopus oocytes. Seven of the assayed compounds were found to inhibit AQP4 function, while three did not. A linear correlation was indicated between the in silico docking energies and the in vitro AQP4 inhibitory activity at 20 lM.  2007 Elsevier Ltd. All rights reserved.

1. Introduction Aquaporin 4 (AQP4) is a member of the aquaporin family of water transporters, and has been shown to be highly concentrated in the astrocytes of mammalian brains, particularly in the perivascular and subpial endfeet.1,2 Since its initial discovery in the brain, the role of AQP4 has been either directly or indirectly implicated in a variety of neurological disease processes, including cerebral edema,3,4 epilepsy,5,6 ischemia,7,8 tumors,9,10 meningitis,11 abscess,12 eclampsia,13 Creutzfeldt–Jakob disease,14 lupus cerebritis,15 lead toxicity,16 and neuromyelitis optica.17 Unfortunately, little is presently known regarding the physiological role of AQP4 in these disease pathologies. O N H

N N S

1 (AZA)

O S O NH 2

N O

S

O S O NH2

2 (EZA)

While the studies described above have uncovered a wealth of information about the roles played by AQP4 under normal and pathological situations, there remains Keywords: Aquaporin; Aquaporin 4; Antiepileptic drugs; Drug design; Xenopus laevis oocytes. * Corresponding author. Tel.: +81 25 227 0677; fax: +81 25 227 0821; e-mail: [email protected] 0968-0896/$ - see front matter  2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2007.12.038

a clear need for developing chemical modulators of that protein, which can be used both in vitro and in vivo. Small molecule modulators are likely to be necessary for elucidating the functional properties of the channel structure in the brain under a variety of conditions and stimuli, as has been proven successful for more typical ion channels. Along these lines, we have identified arylsulfonamide based carbonic anhydrase (CA) inhibitors, including acetazolamide (AZA, 1) and 6-ethoxybenzothiazole-2-sulfonamide (EZA, 2), that are able to inhibit AQP4 mediated water transport in an in vitro functional assay.18 We subsequently realized that certain antiepileptic drugs (AEDs), such as topiramate (TPM, 3) and zonisamide (ZNS, 4), are known to inhibit of a number of CA isozymes19 and also share a number of physiochemical properties with AZA and EZA. That observation, along with previous reports describing the resistance of AQP4 KO mice to the chemoconvulsant pentylenetetrazole,20 prompted us to hypothesize that the antiepileptic properties of AEDs may be in part related with their effects on AQP4. We tested this hypothesis using in silico virtual docking experiments, as well as in vitro functional assays.

2. Results The ligand binding site identified for AZA and EZA, which were shown to inhibit AQP4 water transport,18 was used to study the virtual docking of AEDs 3–16, the results of which are summarized in Table 1. The AEDs studied herein showed a wide range of AQP4 pro-

V. J. Huber et al. / Bioorg. Med. Chem. 17 (2009) 418–424

O

O

O O

O

O

O

3 (TPM)

N H

N O

5 (PHT)

4 (ZNS)

NH2

O

7 (OCZ)

8 (GBP) H N

O

S

N

N

9 (LTG) O

H

N

OH O2N

O

N

Cl

O

Cl

O

NH OH

NH2 N N Cl

NH2

O

O NH

N

CO2H

N

6 (CBZ)

S O

H 2N NH2

O O S O NH2

O S O NH2

O

H N

O N

419

O

NH2

O

2

NH2

CO2H

10 (ESM)

11 (VPA)

12 (TGB)

tein monomer docking energies, approximately 40 kcal/ mol. The most stabilized docking energies among these AEDs were found for TPM and lamotrigine (LTG, 9), 70.7 and 63.7 kcal/mol, respectively, which compared favorably with AZA and EZA, 63.7 and 67.0 kcal/mol, respectively. The least stabilized docking energies were found for ethosuximide (ESM, 10) and valproic acid (VPA, 11), approximately 32 kcal/ mol, and were not indicative of any particular energetic stabilization with the defined binding site. A range of local minima for each of the docked structures, often separated by more than 60 kcal/mol, were identified in the in silico screening of these compounds. Representative examples of the local minima for PHT and LTG are shown in the supplemental material section, Fig. S1. In addition to the observed distribution of docking energies, the AEDs also showed a range of binding geome-

13 (PHB)

15 (LEV)

14 (CPM)

16 (FBM)

tries in and around the active site defined for the model, some of which were considerably different from that of AZA (Fig. 1a, shown for comparison). The presence of the sulfamate group in TPM suggested that this drug might be able to adopt a binding geometry analogous to those of AZA and EZA, in particular where the sulfamate group was aligned in the water channel. However, the lowest energy binding geometries were those where TPM did not enter the pore, but was stabilized primarily by electrostatic and hydrophobic interactions at the mouth of the channel (Fig. 1b). Other AEDs, such as LTG, were able to penetrate the water channel in an apparently energy stabilized manner (Fig. 1c), similar to that of AZA. Relatively large, aromatic AEDs, such as phenytoin (PHT, 5), generally showed an intermediate docking energy and appeared to be unable to enter the channel because of their steric size, but were stabilized in the mouth region (Fig. 1d). Small, non-aromatic

Table 1. Names, docking energies, AQP4 inhibition, and IC50 values for the AEDs studied in this report

a

AED

Common name (abbreviation)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Acetazolamide (AZA) 6-ethoxy-benzothiazole-2-sulfonamide (EZA) Topiramate (TPM) Zonisamide (ZNS) Phenytoin (PHT) Carbamazepine (CBZ) Oxcarbazepine (OCZ) Gabapentin (GBP) Lamotrigine (LTG) Ethosuximide (ESM) Valproic acid (VPA) Tiagabine (TGB) Phenobarbital (PHB) Clonazepam (CPM) Levetiracetam (LEV) Felbamate (FBM) Carbamazepine-10,11-epoxide (CBZ-epoxide) S-(+)-lizcarbazepine (OCZ-OH)

DEdock a

%Inhb

IC50 (IAmax)c

63.7 67.0 70.7 47.5 51.4 45.0d 47.9f 39.0 63.7 33.5 32.3 57.2 42.9 61.8 41.5 57.0 50.2 49.7

78 ± 3 66 ± 4 67 ± 6 48 ± 5 58 ± 4

0.86 (86) 10 (75) 3.3 (48) 9.8 (60)

d,e

33 ± 4 e

54 ± 9

8.1 (64)

e

40 ± 7

40 ± 7

Docking score determined for each compound and ratAQP-4b (kcal/mol). Percent inhibition of AQP4-mediated oocyte swelling at 20 lM. c IC50 (lM) and maximum inhibition (IAmax, %) based on fit of AQP-4 inhibition at various concentrations using a sigmoidal function. d Compare with metabolite 17. e No statistically significant inhibitory effect was observed, P > 0.05. f Compare with metabolite 18. b

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were chosen largely based on their commercial availability, and represented a range of docking energies, binding geometries and general structural features. The assay was completed individually for each AED, where its final concentration was 20 lM in an isotonic medium containing 0.1% DMSO. The hypotonic swelling of hAQP4b injected oocytes incubated in the presence of an AED was compared to those incubated in the presence of a blank (isotonic medium containing 0.1% DMSO), as well as oocytes injected with a water sham, to determine the inhibitory level of that drug (Table 1, Fig. S2). Statistically significant reduction in the hypoosmotic volume change of oocytes incubated in the presence of TPM, ZNS, PHT, OCZ, LTG, and VPA was observed (67%, 48%, 58%, 33%, 54%, and 40% inhibition, respectively, at 20 lM). On the other hand, CBZ, GBP, and ESM had no effect on water permeability at that concentration. Although CBZ showed no inhibitory activity, its metabolite, CBZ-epoxide, showed an inhibitory effect on hAQP4b mediated water transport under the same assay conditions.

Figure 1. Schematic representation of ligand docking to the AQP4 protein monomer. Ligands are shown as space-filling models, while the protein monomer is shown as a ribbon diagram; (a) AZA (1), (b) TPM (3), (c) LTG (9), and (d) PHT (5).

AEDs, such as ESM and VPA, demonstrated no significant affinity for the AQP4 model used in this study. The dibenzoazepine based AEDs, carbamazepine (CBZ, 6) and oxcarbazepine (OCZ, 7) could be considered prodrugs and were known to have metabolites that possess significant anticonvulsant activity.21 These biologically active metabolites, carbamazepine-10,11-epoxide (CBZ-epoxide, 17) and S-(+)-licarbazepine (OCZ-OH, 18), respectively, were also investigated using the AQP4 protein monomer in silico model. Both metabolites showed an increased energetic stabilization relative to their principal compounds. That increase appeared to be significantly greater in the case of CBZ than that of OCZ, dDEdock = 5.2 and 1.8 kcal/mol, respectively. OH

O

N O

N NH2

17 (CBZ-epoxide)

O

NH2

18 (OCZ-OH)

Nine AEDs (3–11) and CBZ-epoxide (17) were then investigated in an in vitro functional assay for modulatory activity against hAQP4b. The selected compounds

The hAQP4b inhibitory activities of TPM, ZNS, PHT, and LTG were further studied in a dose–response experiment. Sample preparation, assay conditions, and procedures were essentially identical to those used for the single point (20 lM) inhibition assay. Percent inhibition was determined at multiple AED concentrations between 0.1 and 100 lM, and the resulting dose–response curves were fit using a sigmoidal function to determine the apparent IC50. Clear dose dependent behavior within the selected concentration range was identified for the four AEDs (Fig. 2). The IC50 values determined for these compounds were quite similar to each other, approximately 10 lM, and were somewhat less potent than that found for AZA (Table 1).18 The maximum inhibitory activity (IAmax) observed for the AEDs seemed to vary between approximately 40–80%, and none of the organic AQP4 inhibitors identified to this point have been able to completely block the water transport. A correlation of the in vitro and in silico assay data was strongly suggested by inspection of the results described in Table 1. In general, compounds with a more strongly stabilized docking energy showed a greater degree of AQP4 inhibition in the functional assay than those less energetically stabilized. Ideally, this correlation would be done using the IC50, Ki or Kd values determined from the biological assay; unfortunately, an insufficient number of IC50 values have been determined to permit such an analysis. However, an apparent Kd value (Kd app) can be defined as Kd app = %Inh/(100 %Inh), which should allow the correlation of the in vitro and in silico results to be studied. Plotting Log (1/Kd app) against DEdock, the in silico docking energy, for the nine compounds shown to possess AQP4 inhibitory activity confirmed the existence of a correlation (Fig. 3). The correlation appeared to be significant, although the goodness of fit was somewhat low, r2 = 0.57. The model used in this study was specific toward the channel, and did not account for any possible allosteric binding sites or other energetic contributions significantly outside of

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potential of AEDs not yet assayed in vitro to be estimated. Hence, those AEDs with a docking energy >50 kcal/mol appeared likely to possess AQP4 inhibitory properties under similar conditions, while those with a score <45 kcal/mol were significantly less likely. The ability to inhibit AQP4 of those AEDs whose docking energy falls between 45 and 50 kcal/mol could not be predicted based on the results presently available. These results strongly suggested that the existing AEDs fall into two groups, specifically those with AQP4 inhibitory activity and those without. 3. Discussion To date, AQP4 has been shown in numerous biological studies to be related to a variety of disease pathologies. However, in many of the studies reported, the role of AQP4 function in the origin and progression of the pathology, and indeed, the degree to which a modulator of AQP4 function could affect it is simply not known. This situation is further exacerbated by the paucity of ligands known to modulate water transport through this protein. Therefore, we believe it is of singular importance to identify AQP4 modulators that can ultimately be used to study this protein’s function in vivo. Figure 2. Dose–response curves for (a) TPM; (b) ZNS; (c) PHT; (d) LTG. Individual points are shown as black squares, error bars represent the total standard error at that point, and solid lines represent the non-linear least-squares fit of the data using a sigmoidal function. IC50 = 10, 3.3, 9.8, and 8.1 lM, respectively.

Figure 3. Correlation of the apparent Kd (Kd app) derived from the in vitro inhibitory activity and the in silico docking energy (DEdock) for the compounds showing AQP4 inhibitory activity (1–5, 7, 9, 11, 17), (j) individual points, (—) linear regression (r2 = 0.57).

the water pore, which may partly account for the low goodness of fit. One potential allosteric site was described as being involved in AQP4 inhibition by the tetraethylammonium cation;22 although, it is not clear if that is the only possible alloseric site. Given the general lack of obvious binding pockets outside of the water channel’s external vestibule, such allosteric binding sites are likely be highly selective, particularly toward small substrates, and difficult to identify. Nevertheless, the degree of correlation observed appeared to be sufficient to allow the AQP4 inhibitory

Within the context of identifying AQP4 modulators that will be useful for in vivo studies, the results presented herein are significant. Arguably, these AEDs will be of little use in directly studying AQP4 function, and any resultant physiological effects of modulating that function. Most of the AEDs lack target selectivity and their physiological effects are well understood. However, based on the docked geometries of the AEDs described in this study, as well as those compounds described previously, a compelling picture of how molecules interact with the AQP4 water channel is beginning to emerge. In particular, a combination of hydrophobic and electrostatic interactions might be necessary to bind at the water channel site. Superimposing the final docked geometries of the AEDs studied in this report reveals a general conservation of binding geometries (Fig. 4). The importance of having both a hydrophobic and electrostatic component can be highlighted by comparing the AQP4 inhibitory activity of CBZ and its metabolite, CBZ-epoxide (17). Both molecules share similar docked conformations; however, the oxo-group in CBZ-epoxide appears to be primarily responsible for the increased stabilization of that ligand versus CBZ (dDEdock = 5.1 kcal/mol). Moreover, many of the other AEDs appear to have similar hydrophobic and hydrogen bond interactions as CBZ-epoxide in their respective docked conformations. The binding geometry of VPA, for which AQP4 inhibition was observed in vitro but for which no stabilized docked geometry can be identified, requires further consideration as there is some possibility that this AED is functional at an allosteric binding pocket. A small hydrophobic pocket spanning two protein monomers in the biological unit may be able to accommodate VPA; although, attempts to dock VPA into this region have not been successful. Indeed, this study has

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effect have yet to be identified, despite a great deal of research on this topic, it may be reasonable to consider the optimization of AQP4 inhibitory activity as another parameter in the development of future antiepileptic agents. 4. Materials and methods The procedures used for the virtual and bio-assays are described in greater detail in the supplemental materials section, and are presented in summary below. 4.1. In silico assay Figure 4. Superposition of the AQP4 docked geometries of AZA (green), TPM (blue), ZNS (yellow), PHT (red), CBZ-epoxide (light gray), GBP (cyan), LTG (orange), ESM (dark gray), and CPM (magenta). AEDs are shown as solid bars and protons have been omitted for clarity. This figure is configured for divergent stereoscopic viewing.

focused on AED binding to the water channel, principally because no binding sites were suggested by the protein structure, or the model subsequently derived from it, that appeared large enough to accommodate most of the AEDs used in this study. Also of significance, these results clearly point to a more direct link between the currently used AEDs and AQP4 modulation. Presently, a number of pharmacological effects are considered to give rise to the antiepileptic properties of various AEDs, these include sodium channel blockade, calcium current inhibition, general GABAergic effect enhancement, and glutamate response desensitization. However, there is no definite agreement regarding the exact mechanisms by which AEDs block epilepsy, and it might instead be argued that some combination of these pharmacological actions play a role in potentiating epileptogenic response. Moreover, a number of antiepileptic therapies have been identified that do not appear to have any direct neurological effect, such as the use of carbonic anhydrase inhibitors, hormones, and a ketogenic diet. The results of this study show that AQP4 inhibition by AEDs may also be part of their antiepileptic effect. The possibility of an AQP4 role in epilepsy has been suggested by various studies. Mice lacking AQP4 showed higher resistance to seizure induction by the chemoconvulsant pentylenetetrazol (PTZ) compared to wild-type mice.20 Impaired water and K+ homeostasis and the misregulation of AQP4 have also been identified in the hippocampus of epileptogenic brains.4,23 Moreover, as a channel structure confined to glia, AQP4 shares a basis in the rapidly expanding concept that glia plays a significant, if not the sole role, in epileptogenesis.24,25 The observed AQP4 inhibition by most of the currently used AEDs represents a previously unknown commonality between them, and appears to be significant because of the different modes of action presumed for these drugs. Therefore, while the necessary pharmacological actions needed to demonstrate an antiepileptic

A protein monomer taken from the rat Aquaporin 4M23 (ratAQP4b) electron diffraction structure26 was imported into the BioMedCAChe molecular modeling environment (Version 6.1.12, Fujitsu Ltd., Tokyo). Protein active site residues were selected primarily from the mouth region of the water channel. The ligand used for the docking simulation was defined using all of the substrate’s atoms, whose structure was independently optimized. The ligand docking was then completed using the Active Site module within BioMedCache and the included PMF energy function. Initial ligand geometries were populated using a genetic algorithm. The active site residue side-chains and ligand were allowed to be flexible during the minimization, while the remainder of the protein atom coordinates were fixed. The final docking energy was reported in Kcal/mol and excluded ligand van der Waals terms. 4.2. In vitro assay All compounds studied in vitro were purchased from Sigma Chemical Corporation (St. Louis) at the highest purity available and were used without further purification. Unless specified, other reagents were purchased from Sigma and Wako Pure Chemical Industries (Osaka) and were used without modification. Modified Barth’s Medium (MBS) and isolation buffer were prepared using autoclaved, doubly distilled water. cDNA encoding the human Aquaporin 4-M23 isoform (hAQP4b) was cloned by reverse-transcription polymerase chain reaction (RT-PCR), and the first-strand cDNA was synthesized from human cerebellum total RNA by using the Advantage RT-for-PCR Kit (CLONTECH). The oligonucleotide PCR primers were designed based on published hAQP-4b sequences.27 Fulllength cRNA was subcloned into pSP35T expression vector for Xenopus oocytes.28 The resulting cRNA was sequenced, and was found to be in agreement with the literature reports.27 Denuded stage V–VI oocytes were prepared from an adult, female Xenopus laevis,29 and were allowed to equilibrate in MBS for a minimum of 2 h at 18 C prior to cRNA injection. A hAQP4b cRNA solution (0.1 lg/ lL cRNA) was prepared from an existing stock solution. An aliquot (30 nL) of either the final cRNA solution or distilled water (sham) was injected into each

V. J. Huber et al. / Bioorg. Med. Chem. 17 (2009) 418–424

oocyte using a Drummond oocyte injection system. The injected oocytes were then incubated for 48 h at 18 C in MBS. Medium was changed and non-viable oocytes removed at 24 and 48 h post injection. Briefly, the oocyte assay procedure, modified from an existing literature report,30 was completed as follows: At least 3 h prior to the assay, oocytes were transferred to a 96-well microplate along with 45 lL of MBS. An aliquot of a 200 lM inhibitor solution (5 lL) or blank (5 lL) was introduced to the oocyte containing well. Following a 2 h incubation period, the assay was performed by diluting the oocyte incubation medium with an aliquot (100 lM) of a buffer-free aqueous solution containing equal concentrations of the inhibitor and DMSO as that in the incubation medium. Oocyte hypotonic expansion was monitored using a digital imaging system, with a nominal magnification factor of 20·. Images were recorded at an interval of 60 s for up to 4 min post dilution. Ooctye images were then transferred to a PC and the area of each oocyte was evaluated using NIH Image-J. The cross-sectional area values for each oocyte at time = t were converted to volumes assuming a spherical relationship. The relative volume of each oocyte compared to its initial volume was then determined. Relative oocyte volumes were then averaged for n oocytes and the standard error was determined at each point. Percent inhibition (%inh) of AQP4 mediated hypoosmotic expansion was given by the relationship %inh = 100(1 {[(Pf ± SE) (Pfsham ± SEsham)]/[(Pfblank ± SEblank) (Pfsham ± SEsham)]}), where Pf ± SE represents the average osmotic permeability of n AQP-4 injected oocytes and their standard error at each inhibitor concentration, Pfsham ± SEsham is similarly the osmotic water permeability of the water injected oocytes, and Pfblank ± SEblank is that of the AQP4 cRNA injected oocytes incubated with a blank solution. The osmotic water permeability (Pf) of each oocyte was determined as described elsewhere.30 Data were evaluated using a single tail ANOVA, P-values less than 0.05 were required to consider the inhibition as having statistical significance. Dose–response analyses were performed as described above; however, the concentration of ligand in both the assay and incubation media was adjusted to 0.1, 1, 5, 10, 20, and 100 lM, respectively, for n = 5 oocytes. Samples were specifically prepared, such that the final DMSO concentration in each assay well was fixed at 0.1% at all concentrations of ligand and the blank solutions. Apparent IC50 values were calculated based on the inhibition values determined from the dose–response data using a sigmoidal function. Acknowledgments Professor Kenji Sakimura, Ms. Atsuko Kitamura, and Ms. Toshie Honma are thanked for their kind assistance. The study was supported by grants from the Ministry of Education, Culture, Sports, Science and

423

Technology (Japan), a Grant for the Promotion of University of Niigata Research Projects (University of Niigata, Center for Transdisciplinary Research), and the Takeda Science Foundation.

Supplementary data Figures included describe the representative examples of local minima structures identified in the in silico screening of 5 and 9, and results of the single dose (20 lM) inhibition assays for compounds 3–11 and 17. Detailed experimental conditions are also presented. Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bmc.2007.12.038.

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