Dipotassium-trioxohydroxytetrafluorotriborate, K₂[B₃O₃F₄OH], is a potent inhibitor of human carbonic anhydrases

Abstract

The boron heterocyclic compound dipotassium-trioxohydroxytetrafluorotriborate (K₂[B₃O₃F₄OH]) was investigated as inhibitor of the zinc enzyme, carbonic anhydrase (CA, EC 4.2.1.1). Eleven human (h) CA isoforms, hCA I–IV, VA, VI, VII, IX and XII–XIV, were included in the investigations. The anion, similar to tetraborate or phenylboronic acid, inhibited most of them. hCA III was not inhibited by K₂[B₃O₃F₄OH], whereas hCA VA, hCA VI, hCA IX and hCA XIII were inhibited in the submillimolar range, with K_Is of 0.31–0.63 mM. hCA I and II (cytosolic, widespread isoforms), hCA IV (membrane-bound isoform), hCA XII (tumor-associated, transmembrane) and hCA XIV (transmembrane) were much more effectively inhibited by this anion, with inhibition constants ranging from 25 to 93 µM. hCA VII, a cytosolic enzyme present in the brain and associated to oxidative stress, was very effectively inhibited by K₂[B₃O₃F₄OH], with a K_I of 8.0 µM. We propose that K₂[B₃O₃F₄OH] binds to the metal ion from the enzyme active site, coordinating to the Zn(II) ion monodentately through its B-OH functionality. We hypothesize that some of the beneficial antitumor effects reported for K₂[B₃O₃F₄OH] may be due to the inhibition of CAs present in skin tumors.

• Anion
• carbonic anhydrase
• dipotassium-trioxohydroxytetrafluorotriborate
• inhibitor

Introduction

Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes, which catalyze the hydration of carbon dioxide to bicarbonate and protons1–3. These enzymes are found in various organisms all over the phylogenetic tree, as five different, genetically distinct families, the α-, β-, γ-, δ- and ζ-CAs1–7. The metal ion from the enzyme active site (which may be Zn(II), Fe(II), Cd(II) or Co(II) among others) is essential for the catalytic activity and also for the binding of most (but not all) classes of CA inhibitors (CAIs) investigated so far, such as the (in)organic anions, the sulfonamides and their isosteres (again as anions), the dithiocarbamates and the xanthates1–12.

Many inorganic anions were investigated extensively for their interaction with such enzymes for obvious reasons: diverse CAs may catalyze the physiologic reaction in the presence of various anions present in the cell, such as chloride, bicarbonate, sulfate, sometimes at high enough concentrations, or in prohibitive pH conditions13–18. Furthermore, some marine/aquatic organisms (which encode CAs belonging to various classes of these enzymes) live in environments rich in salts such as halides, sulfate, carbonate or H₂S, and the interaction of such anions with the enzyme may critically influence their catalytic activity19–22. However, other anions may be useful for the design of potent organic CAIs. Indeed, starting with the discovery of trithiocarbonate () as a weak CAI by this group23, the dithiocarbamates24^,25 and xanthates26 were discovered as highly effective inhibitors, with potential applications as antiglaucoma drugs or antifungals27^,28.

Dipotassium-trioxohydroxytetrafluorotriborate (K₂[B₃O₃F₄OH]) is a boron inorganic derivative prepared by reacting potassium hydrofluoride (KHF₂) with boric acid working in molar ratios of 2:329. The structure shown in Figure 1 has been assigned for this compound29. Recently, the interest in K₂[B₃O₃F₄OH] has been renewed with the report of its usefulness in the prevention and/or treatment of benign/malignant changes of the skin, such as for example, nevus or some forms of skin cancers30–32. K₂[B₃O₃F₄OH] was also shown to possess catalase inhibitory activity33, being hypothesized that the above-mentioned antitumor effects may be due to the inhibition of this enzyme.

Figure 1. Structures of tetraborate (as [B₄O₅(OH)₄]²⁻ ion) (A); phenylboronic acid (B); trioxohydroxytetrafluorotriborate (C) and proposed binding of the last anion to the Zn(II) ion from the α-CA active site (D).

Several CA isoforms, such as CA II, IX and XII are associated with tumors or involved in tumorigenesis and metastatic spread1^,2^,34–39. They have been in fact recently validated as antitumor targets both for the imaging or treatment of hypoxic tumors overexpressing them. The CAIs of the sulfonamide, sulfamate or coumarin type are effective alone or in combination with other anticancer agents in reducing the growth of the primary tumor and the formation of metastases1^,2^,34–39. This is the reason why we decided to investigate whether K₂[B₃O₃F₄OH] may interact with these enzymes.

Materials and methods

Chemistry

K₂[B₃O₃F₄OH] was prepared as reported in the literature29. All other compounds used in this study were commercially available, highest purity reagents, from Sigma-Aldrich (Milan, Italy).

Enzymology

All enzymes used in this work were recombinant ones, obtained as described earlier by our group13^,14.

CA catalytic activity and inhibition assay

An applied photophysics stopped-flow instrument has been used for assaying the CA catalyzed CO₂ hydration activity40. Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 10–20 mM Hepes (pH 7.5) and 20 mM Na₂SO₄ (for maintaining constant the ionic strength), following the initial rates of the CA-catalyzed CO₂ hydration reaction for a period of 10–100 s. The CO₂ concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor concentration, at least six traces of the initial 5–10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of the inhibitor (10 mM) were prepared in distilled–deionized water, and dilutions up to 0.01 µM were done thereafter with distilled–deionized water. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to assay, in order to allow for the formation of the E-I complex. The inhibition constants were obtained by non-linear least-squares methods using PRISM 3 (San Diego, CA), whereas the kinetic parameters for the uninhibited enzymes from Lineweaver–Burk plots, as reported earlier41–44, and represent the mean from at least three different determinations.

Results and discussions

Boronic acids of the type RB(OH)₂ where R may be aryl, aralkyl or arylalkenyl, have been investigated as inhibitors of α-CAs by Winum et al.45. The only other boron-containing inorganic anion, which has been investigated in some detail for its interaction with these enzymes, is tetraborate (), which in fact has the structure depicted in Figure 1(A), as in its hydrated form, it contains the [B₄O₅(OH)₄]²⁻ ion. The structures of K₂[B₃O₃F₄OH] (Figure 1C) as well as that of phenylboronic acid (Figure 1B) also incorporate the B-OH functionalities, which we hypothesized to be responsible for the effective inhibition shown by some arylboronic acids against the CAs45. In fact, although phenylboronic acid itself was shown to be an ineffective, millimolar CAI of most CA isoforms (except CA VII, for which it is an effective inhibitor, with a K_I of 8.3 µM; Table 1)43, many boronic acids incorporating other scaffolds showed low micromolar inhibitory effects against isoforms such as CA I, II, IX and XII45, some of which are important drug targets1–3. Thus, we have investigated the inhibitory properties of K₂[B₃O₃F₄OH] against 11 human CA isoforms, hCA I–IV, VA, VI, VII, IX, XII–XIV (Table 1), comparing its inhibition profile with those of tetraborate and phenylboronic acid.

Table 1. Inhibition constants of boron-containing inhibitors against human α-CAs, for the CO₂ hydration reaction, at 20 °C, pH 7.540.

	KI [mM]†
Inhibitors*	‡	Ph-B(OH)2¶	K2[B3O3F4OH]§
hCA I	0.64	1.56	0.093
hCA II	0.95	1.05	0.084
hCA III	0.69	nt	>50
hCA IV	nt	nt	0.025
hCA VA	nt	1.04	0.55
hCA VI	nt	0.82	0.63
hCA VII	0.68	0.0083	0.008
hCA IX	nt	1.20	0.31
hCA XII	nt	0.80	0.084
hCA XIII	0.65	2.85	0.37
hCA XIV	nt	nt	0.088

*As sodium salt (tetraborate).

†Errors were in the range of 3–5% of the reported values, from three different assays, by a CO₂ hydrase assay method (from three different measurements)40.

nt = not tested.

‡From references13^,43^,44.

¶From reference45.

§This work.

The following should be noted regarding the inhibition data of Table 1:

1. K₂[B₃O₃F₄OH] did not inhibit hCA III, an isoform characterized by a low CO₂ hydrase activity and the presence of a bulky Phe residues (Phe198) in the middle of the active site cavity, which interferes with the binding of bulky inhibitors46. It may be observed that the smaller anion tetraborate showed submillimolar inhibitory activity against hCA III, with a K_I of 0.69 mM (Table 1).

2. Isoforms hCA VA (mitochondrial), hCA VI (secreted in the saliva and milk), hCA IX (transmembrane) and hCA XIII (cytosolic) were inhibited by K₂[B₃O₃F₄OH] in the submillimolar range, with K_Is of 0.31–0.63 mM.

3. Isoforms hCA I and II (cytosolic, widespread enzymes), hCA IV (membrane-bound isoform), hCA XII (tumor-associated, transmembrane) and hCA XIV (transmembrane) were much more effectively inhibited by K₂[B₃O₃F₄OH] compared to the enzymes discussed above, with inhibition constants ranging from 25 to 93 µM.

4. Isoform hCA VII (cytosolic enzyme, present in the brain and associated to oxidative stress)47 was very effectively inhibited by K₂[B₃O₃F₄OH], with a K_I of 8.0 µM. It should be noted that PhB(OH)₂ is also a highly effective inhibitor of this isoform (K_I of 8.3 µM).

Based on the good inhibition data observed against the various human isoforms mentioned above, we speculate that K₂[B₃O₃F₄OH] binds to the metal ion from the enzyme active site, coordinating to the Zn(II) ion monodentately, as presented in Figure 1(D).

Considering the fact that the human skin has many CA isoforms involved in several physiological roles of this organ48, and that in some skin cancers, their expression may be dysregulated, we hypothesize in this study that some of the beneficial antitumor effects reported for K₂[B₃O₃F₄OH] may be due to the inhibition of CAs present in tumors.

Conclusions

We evaluated K₂[B₃O₃F₄OH] as inhibitor of 11 human CA isoforms, hCA I–IV, VA, VI, VII, IX, XII–XIV. The anion similar to tetraborate or phenylboronic acid inhibited most of them. Only hCA III was not inhibited by K₂[B₃O₃F₄OH], whereas hCA VA, hCA VI, hCA IX and hCA XIII were inhibited in the submillimolar range, with K_Is of 0.31–0.63 mM. hCA I and II (cytosolic, widespread isoforms), hCA IV (membrane-bound isoform), hCA XII (tumor-associated, transmembrane) and hCA XIV (transmembrane) were much more effectively inhibited by K₂[B₃O₃F₄OH], with inhibition constants ranging from 25 to 93 µM. hCA VII, a cytosolic enzyme, present in the brain and associated to oxidative stress was very effectively inhibited by K₂[B₃O₃F₄OH], with a K_I of 8.0 µM. We propose that K₂[B₃O₃F₄OH] binds to the metal ion from the enzyme active site, coordinating to the Zn(II) ion monodentately through its B-OH functionality. We hypothesize that some of the beneficial antitumor effects reported for K₂[B₃O₃F₄OH] may be due to the inhibition of CAs present in skin tumors. As boron compounds were also inhibitory against CAs from pathogenic species (bacteria, fungi, etc.), we hypothesize that by inhibiting CAs belonging to other classes, K₂[B₃O₃F₄OH] may also show anti-infective properties49^,50.

Declaration of interest

The authors report no conflict of interest. This work was supported by two EU FP7 research grants (Metoxia and Dynano projects).