The human carbonic anhydrase isoenzymes I and II (hCA I and II) inhibition effects of trimethoxyindane derivatives

Abstract

Carbonic anhydrases (CAs, EC 4.2.1.1) had six genetically distinct families described to date in various organisms. There are 16 known CA isoforms in humans. Human CA isoenzymes I and II (hCA I and hCA II) are ubiquitous cytosolic isoforms. Acetylcholine esterase (AChE. EC 3.1.1.7) is a hydrolase that hydrolyzes the neurotransmitter acetylcholine relaying the signal from the nerve. In this study, some trimethoxyindane derivatives were investigated as inhibitors against the cytosolic hCA I and II isoenzymes, and AChE enzyme. Both hCA isozymes were inhibited by trimethoxyindane derivatives in the low nanomolar range. These compounds were good hCA I inhibitors (Kis in the range of 1.66–4.14 nM) and hCA II inhibitors (Kis of 1.37–3.12 nM) and perfect AChE inhibitors (Kis in the range of 1.87–7.53 nM) compared to acetazolamide as CA inhibitor (Ki: 6.76 nM for hCA I and Ki: 5.85 nM for hCA II) and Tacrine as AChE inhibitor (Ki: 7.64 nM).

• Acetylcholine esterase
• affinity chromatography carbonic anhydrase
• enzyme inhibition
• enzyme purification
• trimethoxyindane derivatives

Introduction

Carbonic anhydrase (CA) enzymes are virtually ubiquitous in all living systems and have a variety of physiological and pathological processes including pH regulation, fluid balance, carboxylation reactions, bone resorption, calcification, glaucoma, cancer, osteoporosis, neurological disorders, tumorigenicity and the synthesis of bicarbonate ()1–5. Carbonic anhydrase catalyzes the reversible hydration of carbon dioxide (CO₂) and water (H₂O) to and a proton (H⁺)6–10.

Carbonic anhydrase is present either in prokaryotes or eukaryotes and are encoded by six distinct evolutionarily non-related gene families: alpha (α), beta (β), gamma (γ), delta (δ), zeta (ζ) and eta (η)-CAs11–13. All human CAs (hCAs) are belonging to the α-class. Up to now, 16 isozymes have been recognized. Among these only 12 are catalytically active (CAs I–IV, CAs VA–VB, CAs VI–VII, CA IX and CAs XII–XIV). Of these CA I, II, III, VII and XIII are cytosolic ones, CA IV, IX, XII and XIV are associated with the cell membrane, CA VA and VB reside in mitochondria, CA VI is secreted in saliva and milk. The CA-related proteins (CARPs) VIII, X and XI resulted without any catalytic activity14–18.

The different CA isoforms widely vary in their inhibition, kinetic properties, pattern of expression in various tissues and cellular localization12^,19. An enzyme inhibitor is a molecule that binds to an enzyme and decreases its activity. An inhibitor can prevent a substrate from entering the active site of the enzyme hindering catalyzing. The inhibition of CA enzymes is crucial for living organism including pathogenesis11^,16. Originally CA inhibitors (CAIs) were clinically used mainly as anti-glaucoma, anticonvulsant agents20, diuretics7 and anti-epileptics, while the novel generation compounds are undergoing clinical investigation as anti-obesity21 or anti-tumor drugs/diagnostic tools6^,11^,22. Additionally, they have recently been used in the management of hypoxic tumors23. It is well known that CAIs bind to a catalytic Zn²⁺ ion in the active site of CA isoenzymes and block their activity10–15. The first aromatic and heterocyclic sulfonamides were clinically used derivatives of acetazolamide24. For regeneration of the basic form of CA, a proton (H⁺) is transferred from the active site to the solvent. This H⁺ transfer may be assisted by active site residues or by buffers present in the medium. The fourth position is occupied by H₂O at acidic pH, and is catalytically inactive. At higher pH, the H₂O molecule binds to Zn²⁺ within the CA active site, and the H⁺ transfer reaction transfers a H⁺ to the solvent, leaving an −OH15^,25.

On the other hand, 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 fibers26. AChE plays a crucial role in the ACh-cycle, including the release of ACh27. Disturbance of cholinergic transmission was found in many clinical and neuropathological studies28^,29. The cholinergic system is strictly dependent on both oxidative metabolism and choline supply18^,30. 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^,31.

Sulfamides are beneficial organic compounds in medicinal chemistry32. They show a wide biological activity spectrum. The sulfamides group (−NH-SO₂-NH−) is present in many organic compounds that are known as potent inhibitors of CA33^,34. Sulfamides bind as anions to the zinc ion (Zn²⁺) in the active site with high affinities for CA isozymes35^,36. The most important classes of hCA inhibitors are aryl-sulfonamides and inorganic anions. The sulfamides head group (−NH-SO₂-NH−) is weakly acidic upon approaching the zinc ion (Zn²⁺). However, it leaves the proton to coordinate Zn²⁺ via electrostatic interactions. The tail of the inhibitor molecule can be substituted by specific functional groups to provide further interactions with the amino acids of hCA37^,38. The synthesis of trimethoxyindane derivatives 1–9 including four sulfamide derivatives (3, 4, 8 and 9) was performed as described previously39.

In this study, we identify the potential inhibition effect of some trimethoxyindane derivatives (1–9) against hCA I, II and AChE, which have crucial effects in medicinal aspects.

Experimental

Carbonic anhydrase isoenzymes were purified by Sepharose-4B-L tyrosine-sulphanilamide affinity chromatography40–43 in accordance to previous studies44. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed after purification of the enzymes. The isoenzyme purities were determined by SDS-PAGE45–49, and a single band was observed for each CA isoenzyme. This method has been described in detail in the previous studies50^,51. For this purpose, acrylamide in the running (10%) and the stacking gel (3%), with SDS (0.1%) were used52–55.

Carbonic anhydrase isoenzyme activities were determined according to Verpoorte et al.56 as described previously57^,58. One unit of enzyme activity was expressed as 1 mol/L of released p-nitrophenol (NP) per minute at 25 °C59. The quantity of protein during enzyme purification was spectrophotometrically determined at 595 nm according to the Bradford method60. Bovine serum albumin was used as the standard protein61–63.

The inhibition effect of trimethoxyindane derivatives (1–9) on CA isoenzymes was measured by the hydrolysis of p-nitrophenyl acetate (NPA) by CA to NP. Then, the amount of NP was recorded spectrophotometrically64^,65. The CA-catalyzed reaction of CO₂ hydration was first observed in the absence of trimethoxyindane derivatives (1–9) and used as a control for the CA isoenzymes.

The inhibition effects of trimethoxyindane derivatives (1–9) against AChE activities were measured according to spectrophotometric method described by Ellman et al.66. Acetylthiocholine iodide (AChI) was used as substrate of the reaction. 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 5,5′-dithio-bis(2-nitro-benzoic)acid (DTNB) with thiocholine, released by the enzymatic hydrolysis of AChI, at a wavelength of 412 nm5^,59.

Activity (%)-[Trimethoxyindane derivative] graphs were drawn and the half maximal inhibitory concentration (IC₅₀) values of each trimethoxyindane derivatives (1–9) exhibiting more than 50% inhibition of CA were calculated after suitable dilutions. IC₅₀ is a measure of the potency of trimethoxyindane derivatives (1–9) in inhibiting CA isoenzyme and AChE enzyme activities. In addition to these values, the K_i values for trimethoxyindane derivatives (1–9) were determined for each enzyme. To determine the K_i values, trimethoxyindane derivatives (1–9) were tested at three different concentrations. K_i is the binding affinity constant of the inhibitor. In these experiments, five different concentrations of substrates were used and Lineweaver–Burk curves were drawn67 in detail as described previously68–71.

Results and discussion

Clinical regulation of the activity of hCA by inhibitors proved to be a reliable therapeutic method for a number of human diseases and for several decades has been a major component of therapy for high blood pressure, glaucoma, hyperthyrosis, hypoglycemia and recently cancer72^,73. It was well known that CAs involved in crucial physiological processes connected with CO₂/ transport and homeostasis, electrolyte secretion in a variety of tissues and organs, biosynthetic reactions including gluconeogenesis, ureagenesis and lipogenesis, respiration, tumorigenicity, calcification and bone resorption36^,74^,75. The active site of hCAs is well conserved in sequence among various isoforms. It has a shape of a deep conical cleft and contains a Zn²⁺ ion with a bound hydroxyl group (Zn²⁺−OH⁻) coordinated by three histidine (His94, His96, His119) residues16^,50.

In the present study, we report the inhibition profiles of trimethoxyindane derivatives (1–9) against the slower cytosolic isoform (hCA I), the more rapid cytosolic isoenzyme (hCA II) and AChE enzyme. The chemical formula of trimethoxyindane derivatives (1–9) was given in Figure 1. Trimethoxyindane derivatives (1–9) demonstrated effective inhibition profile against both CA isoforms and AChE enzyme. When examining the results, the following structure–activity relationship could be easily observed:

1. To describe inhibitory effects, researchers often use an IC₅₀ value. However, a more suitable measure is the K_i constant. K_i values were calculated from Lineweaver-Burk graphs. In this study, both K_i and IC₅₀ parameters of the trimethoxyindane derivatives (1–9) were determined, these values were given (Table 1). As shown in Table 1, the corresponding K_i values were calculated for both CA isoenzyme and AChE enzyme. For the cytosolic isoenzyme hCA I trimethoxyindane derivatives (1–9) had K_i values of ranging in 1.66 ± 0.21–4.14 ± 1.29 nM (Table 1). The most inhibition effect was observed by trimethoxyindane derivatives 3 and 8 with K_i values of 1.66 ± 0.36 nM and 1.66 ± 0.21 nM, respectively. On the other hand, acetazolamide (AZA), used a carbonic anhydrase inhibitor for the medical treatment of glaucoma, idiopathic intracranial hypertension, epileptic seizure, cystinuria, altitude sickness, periodic paralysis, central sleep apnea and dural ectasia, showed K_i value of 6.76 ± 2.55 nM. hCA I is highly abundant in red blood cells and is found in many tissues but its precise physiological function is unknown. CA I is associated with cerebral and retinal edema, and the inhibition of CA I may be a valuable tool for fighting these conditions23^,75.

2. The physiologically predominant cytosolic isoform hCA II is ubiquitous, and is associated with several diseases, including epilepsy, edema, glaucoma and altitude sickness. For the physiologically predominant hCA II, trimethoxyindane derivatives (1–9) had K_i values of ranging in 1.37 ± 0.31–3.12 ± 0.61 nM. On the other hand, AZA, used as a clinical carbonic anhydrase inhibitor showed K_i value of 5.85 ± 2.56 nM. These results clearly shown that all trimethoxyindane derivatives have more than hCA II inhibition effects than that of AZA. Acetazolamide is a well-known example of a clinically established carbonic anhydrase inhibitor76–78 and in recent years we have reported its strong inhibition of both human cytosolic CA I and II. Many studies have shown that the inhibition of hCA II is brought about by an inhibitor’s ability to bind to the catalytic Zn²⁺ in the hCA active site and mimic the tetrahedral transition state7^,15^,25. There are important differences in inhibition between the two isoenzymes (hCA I and II). The main difference in the active site architectures of the two isoenzymes is due to the presence of more histidine residues in the hCA I isoform5^,16. In addition to the Zn²⁺ ligands (His 94, His 96 and His 119) discussed in the “Introduction” section, His 64 of hCA I plays an important role in catalysis. Another important difference between the two isozymes is that CA II contains a histidine cluster, consisting of the following residues: His 64, His 4 His 3, His 10, His 15 and His 17, which is absent in hCA I. Hence, these two isozymes exhibit different affinities for the inhibitors. In general, hCA II has a higher affinity for the inhibitor than hCA I16^,25.

• These extensive studies indicate the importance of CA I and II isoenzyme inhibitors. In our laboratory, CA inhibitory effects are also exhibited by a wide spectrum of phenolic compounds, including melatonin9, capsaicin16, morphine42, vitamin E43, caffeic acid phenethyl ester78, hydroquinone18, antioxidant phenols57, phenolic acids79, rosmarinic acid36, natural product, polyphenols80, dimethoxy-bromophenol derivatives incorporating cyclopropane moieties13, natural phenolic compounds10^,69^,70, antioxidant polyphenol products69^,70, (3,4-dihydroxyphenyl)(2,3,4-trihydroxyphenyl)methanone and its derivatives14, natural and synthetic bromophenols81, carbamates and sulfamoylcarbamates82, sulfonamides derived from dopamine related compounds19, novel sulfonamide derivatives of aminoindanes and aminotetralins8, guaiacol and catechol derivatives17, novel phenolic sulfamides51, novel phenolic benzylamine derivatives6, novel sulfamide analogues of dopamine-related compounds4, novel sulfamides derived from 1-aminoindanes and anilines34, new benzotropone derivatives15, novel benzylsulfamides35, brominated diphenylmethanone and its derivatives64, N-acylsulfonamides50 and novel sulfamides and sulfonamides incorporating a tetralin scaffold5.

1. An acetylcholinesterase inhibitor (AChEI) is a chemical that inhibits the AChE from breaking down acetylcholine (ACh), thereby increasing both the level and duration of action of the neurotransmitter ACh. AChEI have been used in insecticides and nerve gases for chemical warfare83^,84. AChE inhibition effects of trimethoxyindane derivatives (1–9) were determined using commercially available purified AChE (Product no: C3389 - Sigma-Aldrich, St. Louis, MO) from electric eel (Electrophorus electricus) based on the method of Ellman et al.66 AChE was very effectively inhibited by trimethoxyindane derivatives (1–9), with K_i values ranging of 1.87 ± 0.84–7.53 ± 2.91 nM (Table 1). On the other hand, Tacrine, which clinically used as AChE inhibitor for treatment of Alzheimer's disease, had been shown to lower AChE inhibition activity (Ki: 7.64 ± 3.74 nM) than that of all trimethoxyindane derivatives (1–9).

Figure 1. The chemical structures of trimethoxyindane derivatives (1–9) used for carbonic anhydrase isoenzymes (hCA I and II) and acetylcholine esterase enzyme (AChE) inhibition effects.

Table 1. Human carbonic anhydrase isoenzymes (hCA I and II) and acetylcholine esterase enzyme (AChE) inhibition effects of trimethoxyindane derivatives (1–9).

	IC50 (nM)						Ki (nM)
Trimethoxyindane derivatives	hCA I	r^2	hCA II	r^2	AChE	r^2	hCA I	hCA II	AChE
1	3.57	0.9740	3.11	0.9968	5.97	0.9532	4.14 ± 1.29	3.12 ± 0.61	7.53 ± 2.91
2	3.62	0.9922	2.64	0.9588	5.17	0.9367	3.86 ± 1.62	2.02 ± 0.19	7.14 ± 3.22
3	1.84	0.9921	1.44	0.9982	3.11	0.9400	1.66 ± 0.36	1.37 ± 0.31	1.87 ± 0.84
4	3.01	0.9974	1.91	0.9931	4.59	0.9536	2.91 ± 0.71	1.68 ± 0.01	2.83 ± 1.02
5	3.76	0.9935	2.17	0.971	5.68	0.9605	3.21 ± 0.51	1.90 ± 0.43	6.67 ± 2.86
6	2.40	0.9776	1.65	0.9937	3.91	0.9623	2.04 ± 0.60	1.89 ± 0.46	4.67 ± 2.07
7	3.11	0.9814	2.38	0.9953	5.68	0.9617	2.39 ± 0.68	3.11 ± 0.54	3.99 ± 1.43
8	1.89	0.9958	1.39	0.9909	3.17	0.9790	1.66 ± 0.21	1.46 ± 0.21	2.10 ± 0.72
9	2.87	0.9969	2.11	0.9895	4.68	0.9710	2.64 ± 0.53	2.21 ± 0.37	3.39 ± 1.11
AZA*	6.07	0.9154	5.50	0.9636	–	–	6.76 ± 2.55	5.85 ± 2.56	–
Tacrine*	–	–	–	–	7.53	0.9066	–	–	7.64 ± 3.74

*Acetazolamide (AZA) was used as a standard inhibitor for all CAs and Tacrine was used as a standard inhibitor for acetylcholine esterase (AChE) enzyme.

Conclusions

All of trimethoxyindane derivatives (1–9) used in the present study demonstrated effective inhibition profiles against both CA isoforms (hCA I and II) and acetylcholine esterase (AChE) enzyme. These similar inhibition results can be due to high homology between these two CA isoenzymes. Trimethoxyindane derivatives (1–9) were identified potent both CA isoenzymes and AChE enzyme inhibitor. In this study, low nanomolar level of K_i values was observed for each trimethoxyindane derivatives and these compounds can be selective inhibitor of both cytosolic CA isoenzymes and AChE enzyme.

Declaration of interest

The authors declare no conflict of interest. IG and SHE would like to extend their sincere appreciation to the Deanship of Research Chairs Program at King Saud University for funding this research, RGP-VPP-254.