A Rational Approach towards the Development of Human Carbonic Anhydrase Inhibitors as Antiepileptic Agent

Antiepileptic activity study considering the MES model and molecular docking studies were performed for a series of previously synthesized dioxoisoindolin benzene sulfonamide derivatives. The reported molecules were investigated as inhibitors of the zinc metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1), specifically against the hCA I and II isoforms. To get a better insight of these molecules as potential inhibitors we specifically consider the hCA I (Ki values in the range 159 nM to >10000 nM) and hCAII (Ki values in range 1.7 nM to >10000 nM). The most potential molecule explored in the study was 3-(4,5,6,7-tetrachloro-1,3-dioxoisoindolin-2-yl)benzenesulfonamide (Ki=27.7 nM), 3-chloro-4-(4,5,6,7-tetrachloro-1,3-dioxoisoindolin-2-yl)benzenesulfonamide (Ki=4.9 nM) and 4-((4-nitro-1,3-dioxoisoindolin-2-yl)methyl)benzenesulfonamide (Ki=34 nM) respectively with obtained p-value <0.01 in the MES study and showed higher antepileptic activity than acetazolamide (AZM). Moreover a well defined docking score with RMSD value of 1.8 throws light on their effective binding to the active site of both 1AZM and 1ZFQ respectively.


Introduction
Carbonic anhydrases (CAs) are zinc (Zn +2 ) metalloenzymes present in almost all living organism with are five genetically distinct CA families α-, β-, γ-, δ-and ζ-CA's [1][2][3]. Human CAs (hCAs) all belong to the α-family and are present in fifteen isoforms, which differ by molecular features, oligomeric arrangement, cellular localization, distribution in organs and tissues, expression levels, and kinetic properties. CA which catalyze the interconversion between carbon dioxide and bicarbonate and involved in other physiological processes connected with respiration and transport of CO 2 or bicarbonate ion, CO 2 homeostasis, electrolyte secretion in many tissues, biosynthetic reactions, calcification, etc. Inhibition and activation of these enzymes are well understood processes, with most classes of inhibitors binding to the metal centre, and activators binding at the entrance of the active site cavity [1][2][3].
However, because of the large number of hCA isoforms, there is a constant need to improve the inhibition and selectivity profile of the so far developed CAIs, in order to avoid side effects due to inhibition of isoforms not involved in a certain pathology [1][2][3].
Epilepsy is a chronic neurological disorder characterized by seizures generated by the sudden, massive, synchronous excitation of neurons in the brain [6][7][8][9]. Beside the ability to block the voltage Na + -channel, to potentiate GABAergic transmission and to block the kainate/AMPA receptor, TPM occupies a particular place among new anticonvulsants due to its ability to inhibit hCA. The enzyme, catalyzing the interconversion of CO 2 and HCO 3 is quite abundant in the brain, being present in the glia and neurons, mainly as the cytosolic isozymes hCA II, VII and the membrane-bound isoform hCA XIV. Although their function in the brain was not well established, it is known that these proteins are involved in the secretion of cerebrospinal fluid (CSF), being present in the choroid plexus of vertebrates in high amount. Epithelial cells of the choroid plexus possess CA II, III, XII, CA VIII and CA XI ( Figure 2) [8]. It also has been proven that inhibition of the brain hCAs increases the cerebral blood flow with the concomitant raising of the CO 2 partial pressure. hCA inhibition might serve as an anticonvulsant mechanism (at least in some forms of epilepsy), taking into account that the contribution of HCO 3 current to the ionic current through γ-amino butyric acid (GABA A ) receptors on dendrites is increased during periods of high-frequency receptor activation. The excitatory effect of HCO 3 is blocked by membrane permeate CA inhibitors [8,9,[15][16][17].   Enterprises, Kolkata, India and acclimatized in the Institute's Animal House for one week. Animal house had 12:12 hr light and dark cycle (temperature 22 ± 2°C, relative humidity of 30 to 70%) and mice had free access to standard rodent food and tap water.

MES-induced seizures:
The maximal electroshock (MES) is a useful tool for assessing generalized tonic-clonic (grand mal) seizures [21]. Corneal stimulation produces a preferential activation of the forebrain structures, while stimulation through ear-clip electrodes activates the brain stem. In mice, MES-induced seizures consist of initial tonic flexion, then hind limb tonic extension (HLTE), followed by the stage of clonus and terminal stupor [22].
On the day of experimentation animals were randomized into groups of 5 mice each in separate labeled cages for control (0.1% Tween 80), test compounds (100 mg/kg) and standard drugs (AZM and phenytoin; 25 mg/kg). After 30 min of intraperitoneal vehicle or drug administration (10 ml/kg), maximal electroshock was delivered to all animals by a rodent electroconvulsiometer through ear clip electrodes. Seizure was induced by passing alternating current of 45 mA for 0.2 sduration. Animals were observed closely for 2 min for duration and end of HLTE which was the measure of seizure prevention efficacy. The number of animal did not show HLTE and died in a particular group within 24 h was used to calculate percentage protection and percentage mortality, respectively [22].
Statistical analysis: Results were expressed as mean values ± SEM. One-way analysis of variance (ANOVA) was used for statistical comparison followed by post-hoc Newman-Keuls Multiple Comparison test and P<0.05 was considered to be significant difference.  15). It also includes a steric-clash term, adds polar terms featured by Schrodinger to correct electrostatic mismatches [23][24][25][26].

Docking studies
GScore=0.065 × van der Waals energy+0.130 × Coulomb energy+Lipophilic term (Hydrophobic interactions)+H bonding+Metal binding+BuryP (Penalty for buried polar groups)+RotB (Penalty for freezing rotatable bonds)+Site (Polar interactions in the active site) The best docked conformation is similar to the crystal structure conformation of AZM and superimposes with root mean square deviation (RMSD) of 1.5 Å, indicating that GLIDE is reliable software for docking of inhibitors into hCA II.

Anti-epileptic study
The anti-epileptic activity of the synthesized novel hCAIs was studied using mice model of MES-induced seizure at 100 mg/ kg intraperitoneal dose and findings are presented in Table 2. All compounds from tetrabromo-1,3-dioxoisoindolin benzenesulfonamide Generally, the lipophilicity of compounds improve brain penetration of the drug, so that MZA or the lipophilic tert-butoxy carbonyl derivative of acetazolamide tBOC-AZA are more effective as anticonvulsants than acetazolamide, which is very hydrophilic [9].
The high lipophilicity of the best hCA inhibitors was hoped to be propitious with the crossing of the blood-brain barrier. Some of the synthesized sulfonamide derivatives by our groups [18][19][20] (Table 1) showed inhibitory potency against two hCA isozymes(hCA I and II) and expected to be in various important physiological processes, of the same order of magnitude as the clinically used drugs acetazolamide and methazolamide. The anticonvulsant activity of some of the best hCA inhibitors were evaluated in maximal electroshock (MES) method in mice [1,2].  [18][19][20] (data not shown).

Docking studies
Molecular docking of twenty six compounds and other clinically used sulfonamides were performed (Table 1) in order to find out the binding mode analysis (Figure 4). Docking was performed preferably by interest in crystal structure of hCA preferably in 1AZM (hCA I) and 1FZQ (hCA II) [27,28]. All the 'A' chain of the crystal structures catalytic domains of the hCA was considered for docking. The Glide (XP) score of the co-crystalliged ligand AZM is -3.8 to -4.8 and the RMSD values is 1.8 which is considered as good for docking of the ligands ( Table 1). Most of the docking scores of the synthesized compounds are so good enough then standard AZM and other clinically used hCAI.  4 has shown the docked conformations of compound 24, one of the highest docking score compound in catalytic binding pocket of hCA I and II.
The hydrophilic/lipophilic balance has been evaluated by the Schrödinger from Qikprop. QP log Po/w: Predicted octanol /water partition coefficient are with the range limit of -2.0 to 6.5 which predicts the compounds to have good cell permeability.

Conclusions
Antiepileptic activity study considering the MES model and molecular docking studies was performed for a series of previously synthesized dioxoisoindolin benzene sulfonamide derivatives and shown a better insight of these molecules as potential inhibitors we specifically consider the hCA I (Ki values in the range 159 nM to >10000 nM) and hCAII (Ki values in range 1.7 nM to >10000 nM). The most potential molecule explored in the study was 3-(4,5,6,7-tetrachloro-1,3dioxoisoindolin-2-yl)benzenesulfonamide (Ki=27.7 nM), 3-chloro-4-(4,5,6,7-tetrachloro-1,3-dioxoisoindolin-2-yl)benzenesulfonamide (Ki=4.9 nM) and 4-((4-nitro-1,3-dioxoisoindolin-2-yl)methyl) benzenesulfonamide (Ki=34 nM) respectively with obtained p-value <0.01 in the MES study and showed higher antepileptic activity than acetazolamide (AZM). Moreover, the data from anticonvulsant study showed significant protection against MES-induced seizure which further validates a relationship between hCA inhibitors and anti-epileptic activity. Some of the derivatives did not produce marked antiepileptic activity in spite of their CA inhibitor activity. This mismatch could be due to differences in pharmacokinetics as optimum blood-brain permeability is required for any drug to produce therapeutic effect in the central nervous system. The MES seizures induced by transauricular stimulation are more severe and hard to inhibit with anti-epileptic drugs as compared to MES elicited with transcorneal electrodes, due to involvement of the brain stem. However, several compounds in this study abolished all components of generalized tonic-clonic seizures suggesting higher potency that was comparable with MZA and phenytoin. Moreover a well-defined docking score with RMSD value of 1.8 throws light on their effective binding to the active site of both 1AZM and 1ZFQ respectively. The pretreatment time before MES was 30 min for all drugs. Values are the mean ± SEM for five mice. * , ** , *** indicates P<0.05, 0.01, and 0.001 respectively versus control group; ANOVA followed by Newman-Keulsmultiple comparison test for HLTE and Chi-square test for percentage of protection and mortality following MES.