Determination of activities of human carbonic anhydrase II inhibitors from curcumin analogs

Purpose: To evaluate the activities of new curcumin analogs as carbonic anhydrase II (CA-II) inhibitor. Methods: Carbonic anhydrase II (CA-II) inhibition was determined by each ligand capability to inhibit the esterase activity of CA-II using 4-NPA as a substrate in 96-well plates. Dimethyl sulfoxide was used to dissolve each curcumin analog compound, and then diluted with biological buffer. They were then mixed with CA-II solution and to start the reaction, 4-NPA was added. Hydrolysis of the substrate was evaluated at 405 nm after incubation for 2 h at 25 °C. The IC50 value of compounds with inhibitory activity higher than 40 % was then evaluated. Molecular docking was also used to predict enzymeinhibitor interaction. Results: Eight new curcumin analogs were potent to inhibit CA-II activity with IC50 values ranging from 7.92 ± 0.54 to 72.31 ± 2.21 μmol; the lowest value was exhibited by (3E,5E)-3,5-bis[(2hydroxyphenyl)methylidene]piperidin-4-one (a1). Molecular docking analysis revealed that this molecule formed hydrogen bonds with Thr199, Thr200 and Gln92 at the active site of CA-II. Conclusion: These curcumin analogs have inhibitory potential against CA-II; (3E, 5E)-3,5-bis[(2hydroxyphenyl)methylidene]piperidin-4-one (a1) has the highest inhibitory activity and may be useful in the development of CA-II inhibitors for glaucoma treatment.


INTRODUCTION
The general function of carbonic anhydrase (CA) is to catalyze the reversible conversion of carbon dioxide and water to a bicarbonate ion and a proton [1][2][3].Misregulation of this enzyme has been linked to many diseases, such as glaucoma, cancer, and epilepsy [4,5].The isozyme CA-II is known to be one of the most efficient in CO 2 hydration [2].Sulfonamide compounds, which are known as strong inhibitors of CA-II, have been used as commercial drugs to treat glaucoma [6].However, these drugs produce many undesired side effects.Therefore, intensive search for novel drugs is ongoing, through synthesis of new derivatives of known drugs or from new molecular bases.Systematic search for new molecular bases is usually undertaken by exploring compounds from natural products [7,8].
Curcumin and related compounds show promising result as a molecular base on which to design novel inhibitors of CA-II [9].Due to the amounts and the variety of curcuminoids from natural sources are limited, methods of synthesizing curcumin and its analogs have been explored [10].
In the present study, 44 novel curcumin analogs, previously synthesized using microwave irradiation [10], were evaluated for their potential as inhibitors of human CA-II.The basic structure of all 44 curcumin analogs is depicted in Figure 1.

EXPERIMENTAL Materials
All inhibitors were synthesized using microwave irradiation methods, as described in a previous study [10].The side groups of each inhibitor are shown in Table 1.

CA-II inhibition assay
Measurement of the reduction in esterase activity on the substrate p-nitrophenyl acetate (4-NPA) was used to perform CA II inhibition assay [11].The assay was divided into two sections-a pretest and an evaluation test.The former was performed to screen inhibitor molecules that have at least a 40 % inhibitory effect, while the latter was undertaken by measuring IC 50 values for the molecules selected in the previous test.
For comparison, the inhibitory activity of a commercial inhibitor, acetazolamide, which is known as a strong inhibitor of CA-II activity was used as standard [7,12,13].All inhibitor samples were prepared by first dissolving them in dimethyl sulfoxide (DMSO).All inhibitor samples were prepared by first dissolving them in dimethyl sulfoxide (DMSO).To dilute the samples, MOPS buffer at concentration 50 mM (pH 6.9) was used.They were then added with CAII solution.Esterase activity was measured by adding 4-NPA substrate to the mixture and incubating it for 2 h at 25 C.The hydrolysis of the substrate was then evaluated at 405 nm using a Biorad Elisa Reader.The percent inhibition for each sample was calculated as.
where C (i.e., control) = total enzyme activity without inhibitor and T (i.e., test sample) = activity in the presence of test compound.
Inhibitory activity was expressed as IC 50 values (the concentration at which 50 % of the enzyme activity inhibited), which were calculated using Microsoft Excel 2011 from a dose-response curve obtained using at least five concentrations (ranging from 1-10 µg/mL) of the inhibitor and carried out in triplicate.

Molecular docking
The initial structure of CA-II was obtained from the protein data bank (PDB), accession code 2Q38 [14].Redocking LSA503 to the CA-II crystal structure was performed to derive docking parameters that will be used for the docking simulation with curcumin analogs.All ligand structures were prepared by the Marvin Sketch program [15].Molecular docking simulations were observed by Autodock Vina and a genetic algorithm was used for exploring ligand poses inside the active site of CA-II [16].Schematic representations of the ligand poses and interactions were generated by LigPlot programs [17].

Statistical analysis
Measurements were carried out in triplicate and the results presented as mean ± SEM.Statistical analysis was performed by t-test using Microsoft Excel 2011, and p < 0.05 was considered statistically significant.

Molecular docking results
A schematic representation of the binding mode of a1 to CA-II, as found during the molecular docking studies is shown in Figure 2.

DISCUSSION
Based on the above analysis, we observed a correlation between the position of the hydroxyl group and the inhibition potential of the curcumin analogs.The presence of a hydroxyl group at R 2 (ortho position) in a1 molecule apparently cause increasing of inhibition against CA-II compared to the presence of hydroxyl group at R 3 as in a4, b4, and c4; or at R 4 as in a8, b8, and c8.Substitution of the hydroxyl group at the R 2 position with more rigid groups, such as methoxy and benzyl groups, decreased the inhibitory capacity of the molecules.However, compounds c1 and b1 with a hydroxyl group at R 2 exhibited contrasting results with a1, and both molecules showed low levels of inhibition towards CA-II activity.This suggests that the hydroxyl group may not be the only contributor to the inhibitory capacity of the curcumin analogs.Other types of interactions may also play roles that strengthen interactions with the enzyme.
In order to further investigate how the a1 molecule exhibited its strong inhibitory capacity against CA-II activity, we performed a molecular docking simulation using Autodock Vina.The docking result gave insight into the role of some of the functional groups in the a1 molecule that contribute to strengthening its interactions at the molecular level with residues inside the active site of the enzyme.We found that the oxygen atom of the hydroxyl group attached at the R 2 position of a1 strongly interacted via hydrogen bonding with the Thr199 and Thr200 residues that are located near the entrance of the active site cavity.
Another hydrogen bond occurred between the N atom at the R 1 position of a1 and Gln92.We propose that interaction of the N atom at R 1 has a dominant effect on the inhibitory activity of CA-II compared with the hydroxyl interaction at the ortho position.In addition, hydrophobic interactions among nonpolar moieties of the a1 molecule with the nearest nonpolar residues also contributed to stabilizing the position of the a1 molecule inside the CA-II active site.Interestingly, a Zn 2+ ion was not directly involved in the stabilization of the a1 molecule, while most inhibitor molecules, such as the CA-II inhibitor acetazolamide, are strongly bound to this ion [12].
Previously, curcumin has been shown to inhibit CA-II [9].In this study, eight new curcumin analogs also showed to effectively inhibit CA-II at the micromolar range.

CONCLUSION
The findings of this study indicate that curcumin analogs can be used as inhibitors of CA-II.Eight of the compounds demonstrated the potential to inhibit CA-II activity.The most effective inhibitor found in the present study is (3E, 5E)-3,5-bis[(2hydroxyphenyl) methylidene]piperidin-4-one, which is also supported by molecular docking results.This curcumin analog strongly interacts with the residues Thr199, Thr200 and Gln92 in the active site of CA-II.Thus, these findings show facilitate the development of novel CA-II inhibitors for glaucoma treatment.and reproduction in any medium, provided the original work is properly credited.

Figure 1 :
Figure 1: Basic structure of curcumin analogs

Table 1 :
List of ligand molecules

Table 2 :
List of ligand molecules and their inhibitory activity against CA-II

Table 2 :
List of ligand molecules and their inhibitory activity against CA-II

Table 3 :
Inhibition of CA-II with curcumin analogs