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Carbonic anhydrase inhibitors are specific openers of skeletal muscle BK channel of K⁺-deficient rats¹

DOMENICO TRICARICO^*, MARIAGRAZIA BARBIERI^*, ANTONIETTA MELE^*, GIUSEPPE CARBONARA and DIANA CONTE CAMERINO^*,2

^* Unit of Pharmacology, Department of Pharmacobiology, and
Dept. of Medicinal Chemistry, Faculty of Pharmacy, University of Bari, Italy

²Correspondence: Unit of Pharmacology, Dept. of Pharmacobiology, Faculty of Pharmacy, Univ. of Bari, Via Orabona n° 4, 70125 Bari, Italy. E-mail: conte{at}farmbiol.uniba.it

SPECIFIC AIMS

The mechanism of action of carbonic anhydrase (CA) inhibitors responsible for the therapeutic effects in periodic paralysis is currently unknown. CA inhibitors showing varying degrees of CA-inhibition, such as acetazolamide (ACTZ), dichlorphenamide (DCP), hydrochlorthiazide (HCT), etoxzolamide (ETX), methazolamide (MTZ), and bendroflumethiazide (BFT) were selected and tested in vitro on muscle BK channels of K⁺-deficient rats. Effects of these drugs as BK channel openers were compared with their capability to inhibit the CA-enzymes. Patch-clamp and molecular modeling investigations were performed to identify the structural requisites for the CA inhibitors needed to interact and open the muscular BK channel.

PRINCIPAL FINDINGS

1. Activation of BK channel by CA inhibitors
Our work indicates that CA inhibitors activate muscle BK channels in isolated membrane patches by directly affecting BK channel structures. Actions of these drugs on BK channels were observed in the presence of micromolar concentrations of cytosolic free Ca²⁺ ions and at negative voltages. The order of potency as BK channel openers was: ACTZ, DE₅₀=7.3 x 10^–6M > BFT, DE₅₀=5.93 x 10^–5M> ETX, DE₅₀=1.17 x 10^–4M >> DCP (Fig. 1 A). All were capable to fully activate the BK channel with the exception of DCP, which behaves as a partial agonist. In contrast, MTZ and HCT failed to activate the muscle BK channel. The action of these drugs as BK channel openers was not correlated with their capability to inhibit carbonic anhydrase enzymes. Reported efficacies as CA inhibitors = DCP>MTZ>ACTZETX>HCT>>>BFT.

View larger version (17K): [in this window] [in a new window]   Figure 1. Structure-activity relationships of the carbonic anhydrase (CA) inhibitors as BK channel agonists. A) Concentration-response relationships of BK channel currents vs. acetazolamide (ACTZ), dichlorophenamide (DCP), methazolamide (MTZ), etoxzolamide (ETX), hydrochlorthiazide (HCT), and bendroflumethiazide (BFT). Curves were constructed at –60 mV (Vm) in presence of 10 x 10–6 M concentration of free Ca2+ ions in the bath. Drug solutions were applied on the internal side of membrane patches. B) Chemical structures of the CA inhibitors representing lower energy conformers of compounds. Intramolecular hydrogen bonds formed between the hydrogen atom of the imide of the ACTZ or the hydrogen atoms of the sulphonamide moieties of BFT, ETX, MTZ, and DCP. Fluorine, nitrogen, and chlorine atoms are shown. These had calculated inter-atomic distances ranging between 1.82A° and 3.01A°. The HCT molecule formed an intra-molecular hydrogen bond between hydrogen atoms of the sulphonamide moiety and the chlorine atom of the ring (with calculated inter-atomic distances of 3.10A°). All conformers also had a common planar area constituted by an aromatic ring more or less poor of electrons (with the exception of the MTZ molecule). Lack of one of these two pharmacofores led to inactive compounds, as observed for MTZ and HCT molecules, which lost aromaticity and showed an inter-atomic distance of the hydrogen bond >3.01A°.

2. Molecular requisites required for activation of BK channel by CA inhibitors
We showed that the capability of the CA inhibitors to open the muscle BK channel was related to the presence in their chemical structures of an intra-molecular hydrogen bond with calculated inter-atomic distances ranging between 1.82A° and 3.01A° and of an aromatic ring poor of electrons (Fig. 1A, B and Fig. 2 ). The lack of one of these two characteristics led to inactive compounds as observed for MTZ and HCT, which lost aromaticity and showed an inter-atomic distance of the hydrogen bond > 3.01A° (Fig. 1A, B ). Fitting molecules by comparing spatial geometry and electrostatic potential profiles revealed that CA inhibitors showing BK channel opener properties shared common areas of electrostatic potentials with that of the lead compound NS-004. Three negatively charged and one positively charged areas can be identified for the most potent drugs (NS-004, ACTZ, BFT and ETX). These areas represent points of interactions with complementary sites on the receptor and can also play a role in the modulation of channel gating; for example, coordinating and favoring binding of Ca²⁺ ions with Ca²⁺ binding sites on BK channel (Fig. 2) . DCP showed only two negatively charged and one positively charged area, possibly explaining its action as partial agonist of BK channel in our experiments. MTZ and HCT molecules did not fit the electrostatic potential profiles of the active compounds.

View larger version (36K): [in this window] [in a new window]   Figure 2. Schematic Diagram. CA inhibitors according to their structures, act as BK channel openers increasing serum K+-levels and counteracting the symptoms of HYPO-PP such as insulin-dependent fiber depolarization and paralysis. Activation of BK channels by CA inhibitors is caused by interaction of the drugs (in the presence of internal Ca2+ ions) with receptor site/s located in the spatial vicinity of the Ca2+ binding site. Molecules having two pharmacofores such as an aromatic ring poor of electrons (A) and an intramolecular hydrogen bond (B) with interatomic distance ranging between 1.82A° and 3.01A°, interact and form the complex with the receptor site. Additional interactions are also possible with a complementary site on the receptor. R1 = Cl, F, N, and O atoms; R2 = -SO2NH2; -N = COH groups; EWG= electron-withdrawing groups.

CONCLUSIONS

We showed for the first time that CA inhibitors directly activate muscle BK channel in excised patches and this effect is not related to their capability to inhibit carbonic anhydrase enzymes. This mechanism explains the therapeutic effects of these drugs in hypokalaemic periodic paralysis (hypoPP) and possibly in CNS disorders (such as epilepsy) associated to cell depolarization and altered excitability (Fig. 2) . Identification of minimal molecular requisites required for activation of the BK channelby CA inhibitors is critical for screening and selection of drugs targeting the BK channel. The BK channel is the target of several natural and synthetic compounds belonging to the class of cyclic benzimidazolones such as NS-004 and NS-1619. None of these compounds are available on the market for clinical use, while CA inhibitors representing drugs targeting the BK channel and whose effects are Ca²⁺-dependent are available for clinical use. This offers the advantage of limiting drug actions in tissues showing abnormalities of intracellular Ca²⁺ homeostasis (such as in hypoPP) with fewer side effects.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0722fje; doi: 10.1096/fj.03-0722fje

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