The impact of hydroquinone on acetylcholine esterase and certain human carbonic anhydrase isoenzymes (hCA I, II, IX, and XII)

Abstract

Carbonic anhydrases (CAs) are widespread and the most studied members of a great family of metalloenzymes in higher vertebrates including humans. CAs were investigated for their inhibition of all of the catalytically active mammalian isozymes of the Zn²⁺-containing CA, (CA, EC 4.2.1.1). On the other hand, acetylcholinesterase (AChE. EC 3.1.1.7), a serine protease, is responsible for ACh hydrolysis and plays a fundamental role in impulse transmission by terminating the action of the neurotransmitter ACh at the cholinergic synapses and neuromuscular junction. In the present study, the inhibition effect of the hydroquinone (benzene-1,4-diol) on AChE activity was evaluated and effectively inhibited AChE with K_i of 1.22 nM. Also, hydroquinone strongly inhibited some human cytosolic CA isoenzymes (hCA I and II) and tumour-associated transmembrane isoforms (hCA IX, and XII), with K_is in the range between micromolar (415.81 μM) and nanomolar (706.79 nM). The best inhibition was observed in cytosolic CA II.

• Acetylcholinesterase
• carbonic anhydrases
• enzyme inhibition
• hydroquinone
• isoenzymes

Introduction

Acetylcholine (ACh) is synthesized in pre-synaptic terminals from choline and is required for cholinergic neurotransmission in the central and peripheral nervous systems (PNS). In PNS, ACh activates muscles, and is a major neurotransmitter in the autonomic nervous system. Also, ACh induces contraction of skeletal muscle; it acts via a different type of receptor to inhibit contraction of cardiac muscle fibres1^,2. AChE plays a very important role in the ACh-cycle, including the release of ACh3. Disturbance of cholinergic transmission was found in many clinical and neuropathological studies4^,5. The cholinergic system is strictly dependent on both oxidative metabolism and choline supply6. The duration of action of ACh at the synaptic clefts is critically dependent on AChE activity. Recent studies have thrown light on impact of dietary supplementation on the cholinergic system, particularly during aging5^,7^,8.

Due to neuroprotective effects observed in models for Alzheimer’s disease (AD) and Huntington’s disease, a clinical trial revealed a significant improvement of the cognition of AD patients9. However, none of these disease-modifying anti-Alzheimer’s drugs is able to combat or reverse the progression of the disease; they are only able to delay the process, emphasizing the need for more potent AD drugs to modify the disease rather than treating its symptoms only10. AD is a progressive neurodegenerative disease characterized by a loss of cognitive function and behavioural abnormalities. AD is the most common form of dementia11. So far, pathogenesis of AD has not been completely clarified. The only known valid hypothesis being accepted is the lack of enough amount of ACh. Therefore, the compounds that have AChE inhibitory effects were used for the treatment of AD. However, most of these drugs have undesired side effects. Thus, the development and utilization of new effective antioxidants AChE inhibitors are desired10.

Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes present in all life kingdoms, with six genetically distinct families described to date in various organisms12–15. CAs (EC 4.2.1.1) catalyses the simple but crucial reaction between carbon dioxide (CO₂) and water, leading to the formation of protons (H⁺) and bicarbonate 16–19. These enzymes are common to all organisms, from the very simple to complex. This metalloenzyme family is involved in numerous pathological and physiological processes in a different tissues and organs, including biosynthetic reactions, such as gluconeogenesis and lipid and urea synthesis, calcification, lipogenesis, ureagenesis, tumourigenicity, and the growth and virulence of various pathogens and many a lot of physiological or pathological processes such as oedema, glaucoma, obesity, cancer, epilepsy or osteoporosis16–18. Furthermore, six distinct genetic families, the α-, β-, γ-, δ-, ζ- and η-CAs, are known to date, which constituting an interesting example of convergent evolution at the molecular level14^,19–25. These six CA families vary in their preference for the catalytic metal ions used within the active site. These genetically distinct CA families are known to date, (α-, β-, γ-, δ-, ζ- and η-CAs) are metalloenzymes, using Zn²⁺, Cd²⁺, or Fe²⁺ at their active site14^,26–29. The active sites of α-, β-, and δ-CAs contain zinc ions (Zn²⁺), the active sites of γ-CAs have ferrous ions (Fe²⁺) enzymes which activated also with bound Zn²⁺ or Co²⁺ ions. Similarly, Cd²⁺ or Zn²⁺ is found at structure of the ζ-CAs12^,30–32. As the α-isoforms of CA differ in location and tissue distribution, cytosolic (I, II, III, VII, and XIII), membrane-bound (IV, IX, XII, and XIV), mitochondrial (VA and VB), and secreted (VI) forms have been described16. The CA IX and XII isoenzymes are known as the membrane CAs associated with cancers and have also been found in a very limited number of normal tissues, such as gastrointestinal mucosa and related structures16^,33^,34. An important role of CA IX and XII as major tumour prosurvival pH-regulating enzymes was suggested in genetic study35–37.

CA inhibitors (CAIs) are clinically used as anti-glaucoma drugs and diuretics. Additionally, it has lately emerged that CAIs could have potential as anti-cancer, anti-obesity, and anti-infective drugs. Most often, CAIs have been investigated with the classical CAIs belonging to the sulphonamide or sulphamate class, but other chemo-types have also been explored, such as boronic acids, metal-complexing anions and similar small molecules, and phenols38–41. These sulphonamides are clinically used, as anti-glaucoma agents, and also for the therapy of other diseases, such as increased intra-cranial pressure, neuromuscular pathologies, and various neurological disorders, such as epilepsy, hypokalaemia, tardive dyskinesia, genetic haemiplegic migraine and ataxia, essential tremor and Parkinson’s disease, and mountain sickness. For this reason, some chemical compounds and drugs of this pharmacological class are under research and constant development42–44.

Phenolic compounds incorporate a hydroxyl group bonded directly to an aromatic hydrocarbon atom45–52, with the number and position of –OH groups on the aromatic ring creating variety53–58. Hydroquinone (1,4-benzenediol) is significant isomers of phenolic compounds being widely used in medicines, cosmetics, pesticides, flavouring agents, dye and photography59.

In this study, we investigated the effect of hydroquinone on ACh esterase and certain human CA isoenzymes (hCA I, II, IX, and XII).

Results and discussion

Phenols are very active compounds for quenching reactive oxygen species and are reported to possess antioxidant and other anti-cancer, anti-mutagenic, anti-carcinogenic, anti-viral, anti-bacterial, and anti-inflammatory activities60^,61. They are slightly acidic and the phenol molecules have weak tendencies to lose the proton (H⁺) ion from the hydroxyl group (–OH), resulting in the highly water-soluble phenolate anion. Phenolic compounds and polyphenols possess unrelated scaffolds but strong antioxidant properties62^,63. Phenol effectively inhibited CA isoenzymes. The inhibition profile of various isozymes with this class of agents is very variable, with inhibition constants ranging from the millimolar to the submicromolar range for many simple phenols. Antioxidant phenolic compounds inhibit the CA isozymes activity because of the presence of different functional groups in their scaffold, mainly the phenolic –OH and –COOH groups, and the newest class of effective and isoenzyme specific CAIs. In addition to the well-known sulphonamides / sulphamates / sulphamides, it point out to another type of effective CAIs, the phenolic compounds and acids64^,65.

The classical CAIs are the primary sulphonamides, which are in clinical use for more than 50 years as diuretics and systemically acting anti-glaucoma drugs66^,67. Sulphonamide and sulphamates, such as classical CAIs, have long been used clinically as diuretics and anti-glaucoma drugs68–70. In addition, these CAIs exhibit potential anti-cancer, anti-convulsant, anti-infective, anti-pain, and anti-obesity effects. The design of CAIs as therapeutic agents is related to the large number of isoforms in humans, their rather diffuse localization in many tissues or organs, and the lack of isoenzyme selectivity of the presently available inhibitors of sulphonamide or sulphamate types68–73. Indeed, among the sulphonamide derivatives in clinical applications, there are no compounds that selectively inhibit some CA isoforms with therapeutic value16.

Based on studies of CAIs with marked inhibition activity, and considering the literature on phenolic compounds as CAIs74–76, phenols as well as phenolic derivatives constitutes interesting clues for identifying novel CAIs. Here, we report the inhibition profile of the four catalytically active hCA isoforms (hCA I, II, IX, and XII) with hydroquinone. Hydroquinone possesses two phenolic moieties in scaffolds, which are quite variable, and typical of phenolic compounds. We discovered nanomolar to micromolar inhibition of the four hCA isoforms (hCA I, II, IX, and XII).

It was reported that phenolic compounds act as CAIs77–79 by binding to CA in a diverse manner compared to the classical CAIs, which coordinate the Zn²⁺ ion in the active site of the enzyme by substituting a water molecule or hydroxide ion for the fourth, non-protein ligand74^,80. X-ray crystallography studies from Christianson’s group showed the inhibitor to be anchored by means of its –OH moiety to the fourth Zn²⁺ ligand by means of a hydrogen bond. A second hydrogen bond has been evidenced between the oxygen atom of phenol and the amide NH of Thr199, an amino acid residue conserved in all α-CAs16^,33^,81. In the present study, the CA inhibitory ability of hydroquinone as phenolic compounds was measured against the cytosolic isoforms hCA I and II as well as the membrane-associated isoforms hCA IX and XII using the stopped-flow assay method, and the results are displayed in Table 1.

Table 1. Inhibition constants (K_i) of hydroquinone against four acetylcholine esterase enzyme (AChE) and human carbonic anhydrase isoenzymes (hCA I, II, IX and XII).

Enzymes	Hydroquinone
AChE	1.22 nM
hCA I	108.08 μM
hCA II	706.79 nM
hCA IX	309.23 μM
hCA XII	415.81 μM

Hydroquinone exhibited a marked inhibitory activity against cytosolic isoenzyme hCA I with K_i values 108.08 μM (Table 1). On the other hand, acetazolamide (AZA), which used as clinical CAs inhibitor and treatment of glaucoma, altitude sickness, epilepsy, cystinuria, idiopathic intra-cranial hypertension, periodic paralysis, central sleep apnoea, and dural ectasia, had been shown K_i value 184.30 nM). It has been reported that phenolics are not biologically active unless substitution at either the ortho- or para- position has increased the electron density at the –OH group and lowered the oxygen–hydrogen bond energy. As can seen in Figure 1, hydroquinone has two –OH groups at para-position. This position makes hydroquinone highly active in terms of the biological properties. Besides that steric and electronic effects are responsible for biological activity of biomolecules82^,83.

Figure 1. Schematic representation of the proposed interaction between hydroquinone and the active site region of hCA II.

With regard to the profiling assay against hCA II, hydroquinone was highly active, with K_i value 706.79 nM. On the other hand, AZA, which used as clinical CAs inhibitor and treatment of glaucoma, had been shown K_i value 61.10 nM. The result clearly showed that hydroquinone is the highly suitable inhibitor for cytosolic isoenzymes hCA II. The schematic representation of the proposed interaction between hydroquinone and the active site region of hCA II is presented in Figure 1. In hydroquinone, two identical hydroxyl groups (–OH) are bonded to a benzene ring in an para-position. It was reported that para-position is favour of biological activity12^,82^,83. Many studies have demonstrated that the inhibition of CA II is due to the ability of an inhibitor to mimic the tetrahedral transition state when binding to the catalytic Zn²⁺ located in the active site78^,84. CA II protein fold, Zn²⁺ ion, and its coordination by three histidine residues22. It was reported that phenols, which bind by interacting with a water molecule/hydroxide ion coordinated to Zn²⁺ through hydrogen bonding85. The physiologically dominant cytosolic isoform hCA II is ubiquitous and is being involved in several diseases, such as epilepsy, oedema, glaucoma, and altitude sickness86.

Up to now, 16 isoforms of hCA have been discovered; among them the dimeric transmembrane glycoproteins hCA IX and XII are also human-associated CA isoforms having extracellular active site and are marker for a broad spectrum of hypoxic tumour types22^,85. Many sulphonamide derivatives have been investigated for their CA inhibition activity in the search for selective hCA IX and hCA XII inhibitors because their lack of selectivity is the major challenge for the wide use of chemotherapeutic agents in cancer therapy87–92. Both CA IX and XII are overexpressed in many such tumours in response to the hypoxia inducible factor pathway, and research on the involvement of these isozymes in cancer has progressed significantly in recent years85^,93.

Regarding the hCA IX isoenzyme, the tested phenolic hydroquinone compound showed moderate inhibition activity with inhibition constant of 309.23 µM. hCA IX is a catalytically active plasma membrane isoform of CA that normally controls the differentiation of gastric mucosa. Otherwise AZA had been shown K_i value 61.10 µM. Its abnormal expression is strongly associated with tumours, and it is often regulated by hypoxia. Indeed, the expression level of hCA IX was elevated in response to hypoxia, which is a consequence of the rapid growth of many tumours. Considering the abnormally high expression of CA IX in many hypoxic tumours and its demonstrated role in the tumour acidification processes and oncogenesis, this isoform constitutes an attractive target for anti-cancer therapy94.

Finally, potent inhibition was exhibited by hydroquinone against the second tumour-associated isoenzyme hCA XII. As in the tumour-associated isozymes hCA IX, hCA XII were also effectively inhibited by hydroquinone, with K_i value of 415.81 μM against the hCA XII. Also, AZA, a positive standard, had been shown K_i value 0.006 µM. The results showed that inhibition profile of hydroquinone for the transmembrane, tumour-associated isoform hCA IX was rather similar to that for another transmembrane, tumour-associated isoform hCA XII. It has been reported that the biological activity of phenolics depends on the number and position of the –OH groups bound to the aromatic ring, the binding site and mutual position of –OH in the aromatic ring, and the type of substituents45^,94^,95.

Inhibition of AChE enzyme was determined on commercially available purified AChE (Product no: C3389-Sigma-Aldrich, St. Louis, MO) from electric gel (Electrophorus electricus) based on the method of Ellman et al.96 Also, it was demonstrated that the main AChE inhibitory effects was primarily associated with aromatic compounds and, to lesser degree, with aliphatic compounds97. AChE was very effectively inhibited by hydroquinone, with K_i value of 1.22 nM (Table 1). K_i value of hydroquinone for AChE was calculated from Lineweaver–Burk plots (Figure 2). On the other hand, donepezil hydrochloride, which is used for the treatment of mild-to-moderate AD and various other memory impairments, had been shown to lower AChE inhibition activity (IC₅₀: 55 nM)98. Donepezil hydrochloride had N-benzylpiperidine and an indanone moiety that shows longer and more selective action.

Figure 2. Determination of K_i value of hydroquinone for acetylcholinesterase (AChE) enzyme by Lineweaver–Burk plots.

Conclusion

To effect of hydroquinone against hCA I, II, IX, XII and AChE was evaluated. It effectively inhibited hCA I, II, IX, XII isoenzymes, with K_i values in the range of 309, 23–415.8 µM. The various isozymes showed diverse inhibition profiles. These data may explain the beneficial health effects of some of these hydroquinone and may lead to drug design campaigns for more effective CAI. Also, AChE strongly inhibited hydroquinone with K_i of 1.22 nM.

Determination of hCA activity

An Applied Photophysics stopped-flow instrument was used to assay the catalytic/inhibition of various CA isozymes, as reported by Khalifah99. Phenol Red (20 mM) was used as an indicator, with an absorbance maximum of 557 nm, with HEPES (10 mM, pH 7.4) as a buffer and 0.1 M Na₂SO₄ or NaClO₄ (for maintaining constant the ionic strength; these anions are not inhibitory at the used concentration). The CA-catalysed CO₂ hydration was followed for a period of 10–100 s.

Determination of hCA isoenzymes inhibition

For the determination of the kinetic parameters and inhibition constants, the saturated CO₂ concentrations ranged from 1.7 to 17 mM. For hydroquinone, at least six traces of the initial 5–10% of the reaction were used for determining the initial velocity. The uncatalysed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (10 mM) were prepared in distilled-deionized water, and dilutions up to 0.01 µM were performed with distilled-deionized water. Hydroquinone and enzyme solutions were pre-incubated together for 15 min at room temperature prior to the assay to allow for the formation of the E–I complex. The inhibition constant of hydroquinone was obtained by non-linear least-squares methods using PRISM 3, as reported earlier, and represents the mean from at least three different determinations. Human CA isozymes were prepared in recombinant form as reported earlier by our group62^,63^,100^,101.

Determination of acetylcholine esterase activity

The inhibitory effect hydroquinone on AChE activities were measured according to spectrophotometric method of Ellman et al.96 Acetylthiocholine iodide (AChI) was used as substrate of the reaction. 5,5′-dithio-bis(2-nitro-benzoic)acid (DTNB, D8130-1 G, Sigma-Aldrich) was used for the measurement of the AChE activity. Briefly, 100 mL of Tris/HCl buffer (1 M, pH 8.0), 10 mL of sample solution dissolved in deionized water at different concentrations and 50 mL AChE (5.32 10⁻³ U) solution were mixed and incubated for 10 min at 25 °C. Then, 50 mL of DTNB (0.5 mM) was added. The reaction was then initiated by the addition of 50 mL of AChI (10 mM, product no: 01480-1 G, Sigma-Aldrich). The hydrolysis of these substrates was monitored spectrophotometrically by the formation of yellow 5-thio-2-nitrobenzoate anion as the result of the reaction of DTNB with thiocholine, released by the enzymatic hydrolysis of AChI, at a wavelength of 412 nm102.

Determination of acetylcholine esterase inhibition

In order to determine the effect of hydroquinone on AChE, different hydroquinone concentrations were added into the reaction medium. The enzyme activity was measured, and an experiment in the absence of drug was used as control. The IC₅₀ value was obtained from activity (%) versus hydroquinone concentration plots. To determine the K_i constant in the media with hydroquinone as inhibitor, the different substrate (ACh) concentrations were used. Inhibitor solution was added into the reaction medium, resulting in three different fixed concentrations of inhibitor. Lineweaver–Burk graphs103 were drawn using 1/V versus 1/[S] values. K_i constant was calculated from these graphs. Donepezil hydrochloride was used as a reference compound.

Declaration of interest

This work was financed in part by two EU projects of the 7th FP, Metoxia and Dynano. Also, IG and SE would like to extend his sincere appreciation to the Deanship of Scientific Research at King Saud University for funding this research, RGP-VPP-254.

The authors have declared no conflict of interest.