Carbonic anhydrase inhibitors: inhibition of human and bovine isoenzymes by benzenesulphonamides, cyclitols and phenolic compounds

Abstract

Carbonic anhydrase inhibitors (CAIs) are a class of pharmaceuticals used as anti-glaucoma agents, diuretics and anti-epileptics. We report here the inhibitory capacities of benzenesulphonamides, cyclitols and phenolic compounds 1–11 against three human CA isozymes (hCA I, hCA II and hCA VI) and bovine skeletal muscle carbonic anhydrase III (bCA III). The four isozymes showed quite diverse inhibition profiles with K_i values ranging from low micromolar to millimolar concentrations against all isoenzymes. Compound 5 and 6 had more powerful inhibitory action against hCA I and very similar action against hCA II and hCA VI as compared with acetazolamide (AZA) and sulphapyridine (SPD), specific CAIs. Probably the inhibition mechanism of the tested compounds is distinct of the sulphonamides with RSO₂NH₂ groups and similar to that of the coumarins/lacosamide, i.e. binding to a distinct part of the active site than that where sulphonamides bind. These data may lead to drug design campaigns of effective CAIs possessing a diverse inhibition mechanism compared to other sulphonamide/sulphamate inhibitors.

Keywords::

• Carbonic anhydrase
• inhibition
• glaucoma
• cyclitol
• benzenesulphonamide

Introduction

The carbonic anhydrases (CAs, EC. 4.2.1.1) represent a class of ubiquitous zinc-containing enzymes widespread in the all living organisms, which classically participate in the maintenance of pH homeostasis in mammalians, catalysing the reversible hydration of CO₂ in a two-step reaction to yield HCO₃⁻ and H⁺ (see Ref. 1). At least 16 CA isozymes have been described up to now in mammals, the most active ones as catalysts for carbon dioxide hydration being CA II and CA IX^2–5. The CA II is found primarily in red blood cells but also in many other tissues, such as the kidney, lung, eye, etc.^1–4 The CA VI is a secretory isoform, which was initially described, in the salivary glands, saliva, milk and normal human serum³. Other CA isoforms are found in a variety of tissues where they participate in several important biological processes such as acid–base balance, respiration, carbon dioxide and ion transport, bone resorption, lipogenesis and electrolyte secretion^1–12.

Due to the important roles of CAs in higher vertebrates, compounds possessing CA inhibitory properties, mainly aromatic/heterocyclic sulphonamides (such as sulphapyridine (SPD) and acetazolamide (AZA) (Figure 1) have been used for more than 50 years as drugs in the therapy of different pathologies, such as anti-bacterial, glaucoma, various neurological/neuromuscular disorders, epilepsy, acid–base disequilibria or as diuretics^1–5. A class of derivatives which showed very promising applications among the various CAIs, reported by Supuran’s group in the last years, were the thioureas obtained from isothiocyanato sulphonamides (such as, e.g. 4-isothiocyanatobenzenesulphonamide) and amines, hydrazines or amino acids^13–20. Such compounds generally showed potent inhibitory activity against the cytosolic isozyme hCA II as well as the trans-membrane, tumour-associated isozyme hCA IX, being thus interesting candidates for developing anti-glaucoma/anti-tumour therapies based on them^13–18. Many sulphonamide derivatives have been widely used as pro-drugs or drugs. For instance, sulphadiazine is used as an antibiotic, SPD is mainly used for treatment of bacterial infections and AZA is mainly used as an anti-glaucoma agent.

Figure 1.  Chemical structures of commonly used medical sulphonamides.

This family of pharmacological agents takes advantage of the sulphonamide moiety as anchoring group to coordinate the zinc ion within the active site of the enzyme, leading to ligands with micro-nanomolar affinity^6–11.

In the present study we have purified CA I, II, VI (hCA I, hCA II and hCA VI) from fresh human blood and CA III from bovine skeletal muscle tissue (bCA III)¹² and examined the in vitro inhibition effects of compounds 1–11 mentioned above on these enzymes, using the esterase activity with 4-nitrophenylacetate (NPA) as substrate.

Materials and methods

The CNBr-activated Sepharose 4B, protein assay reagents, p-aminobenzene sulphonamide L-tyrosine, 4-NPA and chemicals for electrophoresis were purchased from Sigma-Aldrich Co. All other chemicals were of analytical grade and obtained from either Sigma or Merck.

CA purification assay

The purification of the two CA isozymes was performed with a simple one step method by a Sepharose-4B-aniline-sulfanilamide affinity column chromatoghrapy¹¹. The hCA I was purified, 108.4-fold with a specific activity of 935.42 EUmg⁻¹ and overall yield of 72.42%; hCA II was purified, 822.71-fold with a specific activity of 7100 EUmg⁻¹ and overall yield of 83.98%; and hCA VI was purified, 81.7-fold with a specific activity of 242 EUmg⁻¹ and overall yield of 18.2%^10–14.

CA activity assay and kinetic studies

The CA activity was assayed by following the change in absorbance at 348 nm of 4-NPA to 4-nitrophenylate ion over a period of 3 min at 25°C using a spectrophotometer (Shimadzu UV-VIS) according to the method described by Verpoorte et al.¹⁶ The enzymatic reaction, in a total volume of 3.0 mL, contained 1.4 mL 0.05M Tris-SO₄ buffer (pH 7.4), 1mL 3 mM 4-NPA, 0.5 mL H₂O and 0.1 mL enzyme solution. A reference measurement was obtained by preparing the same cuvette without enzyme solution. The inhibitory effects of the sulphonamide derivatives were examined. All compounds were tested in triplicate at each concentration used. Different concentrations of the compounds were used. Control cuvette activity in the absence of inhibitor was taken as 100%. For the compounds, an Activity (%)-[Inhibitor] graphs were drawn. In these experiments, 4-NPA was used as substrate at five different concentrations (0.15–0.75 mM).

Inhibitory effects of the compounds 1–11 on enzyme activities were tested under in vitro conditions; K_i values were calculated from Lineweaver–Burk graphs and are given in Table 1^15,16.

Table 1.  The hCA I, II, VI and bCA III inhibition data with compounds 1–11, SPD and AZA.

Compound	Ki (μM)
	hCA I	hCA II	bCA III	hCA VI
1	50.11	1.03	2.43	24.30
2	210.5	14.34	13.14	13.46
3	70.17	3.72	2.67	4.48
4	378.9	21.7	57.4	275.3
5	5.481	0.47	0.59	1.12
6	9.336	0.82	1.75	3.78
7	21.72	1.84	1.19	16.73
8	134.6	4.72	4.28	76.24
9	10.2^a	5.5^a	4.63	208^a
10	4003^a	9.9^a	5.79	606^a
11	7.41^a	0.54^a	66.73	0.52^a
SPD	26.23	0.348	93.16	11.67
AZA	36.22	0.374	263	0.348

Mean from at least three determinations. Errors in the range of 1%–3% of the reported value (data not shown); ^afrom Ref. 25.

Protein determination

Protein quantity was determined spectro-photometrically at 595 nm according to the Bradford method during the purification steps, using bovine serum albumin as the standard¹⁷.

Sodium dodecyl sulphate polyacrylamide gel electrophoresis

The sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis was performed after purification of the enzymes. It was carried out in 10% and 3% acrylamide for the running and the stacking gel, respectively, containing 0.1% SDS according to Laemmli procedure¹⁸.

Results and discussion

We report here the first study on the inhibitory effects of benzenesulphonamides, cyclitols and phenolic compounds of type 1–11 on the esterase activity of hCA I, II, VI and bCA III. The sulphonamide carbonic anhydrase inhibitor (CAI), AZA and SPD have been used as negative controls in our experiments, and for comparison reasons. Data of Table 1 show the following regarding inhibition of hCA I, II, VI and bCA III with compounds 1–11, by an esterase assay, with 4-NPA as substrate:

1. Against the slow cytosolic isozyme hCA I, compounds 2, 4, and 10 behave as weak inhibitors, with K_i values in the range of 0.215–4 mM^20–25. Catechol 10 was an ineffective hCA I inhibitor (K_i of 4003 μM). A second group of derivatives compounds 1, 3, and 8 showed better inhibitory activity as compared to the previously mentioned compounds, with K_i values of 50.11–134.6 μM (Table 1). Therefore, the nature of the groups in ortho-, para- and meta- positions strongly influences hCA I inhibitory activity. It is also interesting to note that pyrogallol, (1R,2S)-cyclohexane-1,2-diol, benzenesulphonamide derivatives 5 and 6 were much better hCA I inhibitors as compared to the corresponding other compounds. The AZA and SPD are also medium inhibitors with this assay and substrates against hCA I (K_i-s of 26.23 and 36.22 μM, respectively). Kinetic investigations (Lineweaver–Burk plots, data not shown) indicate that similarly to sulphonamides and inorganic anions [22-25], all the investigated compounds act as non-competitive inhibitors with 4-NPA as substrate, i.e. they bind in different regions of the active site cavity as compared to the substrate. However, the binding site of 4-NPA itself is unknown, but it is presumed to be in the same region as that of CO₂, the physiological substrate of this enzyme¹³.

2. All compounds except for 2 and 4 had better inhibitory activity against the rapid cytosolic isozyme hCA II (Table 1). Compounds 5 and 6 showed the most powerful hCA II inhibitory activity, similar to specific CAI AZA and SPD, with K_i-s of 0.47 and 0.82 μM, respectively (Table 1), whereas the compounds 2 and 4 were weak hCA II inhibitors, with K_i-s of 14.34 and 21.72 μM, respectively (Table 1). Structure–activity relationship (SAR) is thus quite sharp for this small series of mono-, di-, tri-hydroxy compounds. The best hCA II inhibitor in this series of derivatives 5 was the bulky, which with a K_i of 0.47 μM is similar inhibitor AZA and SPD, a clinically used sulphonamide. It must be stressed that K_i-s measured with the esterase method are always in the micromolar range because hCA I and II are weak esterases^{10,11,21–24}.

3. Compound 5–7 are also strong inhibitors of bCA III, with K_i-s in the range of 0.59–1.75 μM. However, again compounds 2 and 4 are weak inhibitors (K_i of 13.14–57.4 μM). Other compounds were determined to be medium potency inhibitors (K_i-s of 3.78–24.3 μM) (Table 1).

4. Compounds 4 and 8–10 are also weak inhibitors of the secreted isozyme hCA VI, with K_i-s of 76.24–606 μM. However, again the compounds 1–3, 6 and 7 are medium potency inhibitors (K_i-s of 3.78–24.3 μM), and compounds 5 and 11 show a higher affinity for this isozyme, with inhibition constant in the ranges of 0.52–1.12 μM (Table 1).

Although various CAIs have been identified, it is critically important to explore further classes of potent CAIs in order to detect compounds with a different inhibition profile to find novel applications for the inhibitors of these widespread enzymes.

Especially, compound 5 4-Methyl-N-((1S,2R,3S,6S)-2,3,6-trihydroxycyclohexyl) benzenesulphonamide influence the activity of hCA and bCA isozymes due to the presence of the OH groups bound to the sulphonamide moiety. Compound 6 4-Methyl-N-((1S,2R,3R,6S)-2,3,6-trihydroxycyclohexyl) benzenesulphonamide shows relatively lower action although it has the same structure except for the configuration of one hydroxyl group. Thus, the nature of the substituents strongly influences the inhibitory potency of these molecules²⁶. Our findings indicate, thus, another class of possible CAIs of interest, in addition to the well-known sulphonamides/sulphamates/sulphamides, the phenols/biphenyl diphenols bearing bulky ortho-moieties in their molecules. Some of the compounds investigated here showed effective CA inhibitory activity, in the low micromolar range, by the esterase method which usually gives K_i-s an order of magnitude higher as compared to the CO₂ hydrase assay²⁵. Probably the inhibition mechanism of these compounds is distinct of the sulphonamides with RSO₂NH₂ groups and similar to that of the coumarins/lacosamide, i.e. binding to a distinct part of the active site than that where sulphonamides bind. These findings point out that substituted benzenesulphonamide compounds may be used as leads for generating potent CAIs eventually targeting other isoforms which have not been assayed yet for their interactions with such agents.

Figure 2.  Chemical structures of compounds 1–11.

Declaration of interest

The authors greatly acknowledge the Scientific and Technical Research Council of Turkey (TUBITAK) for financial support (Project No: 106T374) for (INK). This study was financed by Turkish Republic Prime Ministry State Planning Organization (DPT), (Project no: 2010K120440) for (MS).