Inhibition of Aquaporin 4 by antiepileptic drugs

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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 μM.

Graphical abstract

Several clinically approved antiepileptic drugs, such as phenytoin, lamotrigine, and topiramate, were found to inhibit the water transport properties of Aquaporin 4 in an in vitro functional assay.

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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.

While the studies described above have uncovered a wealth of information about the roles played by AQP4 under normal and pathological situations, there remains 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.

Section snippets

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 316, the results of which are summarized in Table 1. The AEDs studied herein showed a wide range of AQP4 protein 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,

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

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.

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 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.

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