Recent advances in the discovery of zinc-binding motifs for the development of carbonic anhydrase inhibitors

Abstract

In addition to the sulfonamides and their isosteres, recently novel carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs) which act by binding to the metal ion from the active site were discovered. Based on the X-ray crystal structure of the CA II–trithiocarbonate adduct, dithiocarbamates, xanthates and thioxanthates were shown to potently inhibit α- and β-CAs. The hydroxamates constitute another class of recently studied CAIs both against mammalian and protozoan enzymes. Another chemotype for which CA inhibitory properties were recently reported is the salicylaldoxime scaffold. X-ray crystal structures were reported for CA II complexed with dithiocarbamates and hydroxamates, whereas the xanthates and salicylaldoximes were investigated by kinetic measurements and docking studies. The dithiocarbamates and the xanthates showed potent antiglaucoma activity in animal models of the disease whereas some hydroxamates inhibited the growth of Trypanosoma cruzii probably by inhibiting the protozoan CA.

• Carbonic anhydrase
• inhibitors
• zinc-binding motif

Introduction

The zinc metalloenzyme carbonic anhydrases (CA, EC 4.2.1.1) catalyze a very simple but essential physiological reaction, carbon dioxide hydration to bicarbonate and protons. These enzymes belong to a superfamily of ubiquitous zinc proteins which are involved in many important physiologic processes in all life kingdoms (Bacteria, Archaea, and Eukarya). Five genetically distinct enzyme families are known to date: the α-, β-, γ-, δ-, and ζ-CAs. All of them are metalloenzymes, but α-, β-, and δ-CAs use Zn(II) ions at the active site, the γ-CAs are probably Fe(II) enzymes (but they are active also with bound Zn(II) or Co(II) ions), whereas the ζ-class uses Cd(II) or Zn(II) to perform the physiologic reaction catalysis. The catalytic mechanism of the CAs is well understood, the metal ion being essential for the catalytic process1. Inhibitors of these enzymes (CAIs) started to be investigated more than 60 years ago, and found pharmacological and clinical applications in the field of anti-glaucoma, anti-convulsant, diuretics and recently as anti-cancer agents. Such CAIs target diverse isozymes of the 13 catalytically active alpha-CA isoforms present in humans. Moreover, it has also emerged in the past four years that these enzymes can be used as potential target for designing anti-infective drugs with a novel mechanism of action. Diverse studies were ultimately reported during the past few years for some pathogenic protozoa (Plasmodium falciparum), fungi (Cryptococcus neoformans, Candida albicans, Candida glabrata, and Saccharomyces cerevisiae), and bacteria (Helicobacter pylori, Mycobacterium tuberculosis, and Brucella suis)2^,3.

From earlier studies, three major basic structural elements have emerged to be important in the design of CAIs. The first one is the zinc-binding function (ZBF) that interacts with the active site metal ion and the residues Thr199 and Glu106. The second one is the organic scaffold, usually an aromatic or heteroaromatic moiety, which interacts both with the hydrophobic as well as the hydrophilic halves of the active site. The third one is a ‘‘tail’’ attached to the organic scaffold4^,5.

ZBFs constitute a critical structural parameter for the design of novel classes of selective CAIs, and variation of the nature of this motif is necessary to identify compounds with a diverse inhibition profile as compared to the clinically used drugs which generally indiscriminately inhibit many CA isoforms, thus leading to various side effects. Sulfonamides and their isosteres (sulfamates/sulfamides) constitute the main class of CAIs which bind to the zinc metal ion within the enzyme active site. Even if the classical sulfonamide function still constitutes the main motif used in the design of carbonic anhydrases inhibitors, new zinc-binding chemotypes have recently emerged leading to promising new classes of compounds that inhibit CAs from human or from bacterial and fungal pathogens6–9.

In this paper, we will compile the last developments in the field of zinc-binding motifs reported the past four years in the design of carbonic anhydrase inhibitors.

Dithiocarbamates and derivatives

In the past four years, dithiocarbamate (DTC) moiety emerged as a new important chemotype for the design of carbonic anhydrase inhibitors. This new zinc-binding group was rationally discovered after the report that inorganic anion trithiocarbonate was able to inhibit carbonic anhydrase isoforms in the milli-micromolar range. X-ray crystal structure of this inorganic anion bound to CA II was also reported showing that trithiocarbonate was monodentately bound to the Zn(II) ion making several hydrogen bonds with Thr199 and two water molecules from the enzyme active site (Figure 1)10^,11.

Figure 1. Structure of trithiocarbonate and dithiocarbamate zinc-binding motif. Electron density of trithiocarbonate (yellow and gold) bound within the hCA II active site. The electron density of the two water molecules (w162 and w179) making hydrogen bonds with the bound inhibitor are also presented. The catalytically critical Zn(II) ion is shown as the violet sphere, and its three protein ligands, His94, His96 and His119 in CPK colors. Thr199, a conserved amino acid residue involved in catalysis and binding of inhibitors is also shown, whereas the protein backbone is in green.

Thus, the was detected as a new zinc-binding motif and a rather large series of such compounds have been reported for their inhibitory activity against human CAs. In 2012, Carta et al. described a large series of 27 DTCs. These compounds were tested for their inhibitory activity against four human (h) isoforms hCA I, II, IX and XII. Numerous inhibitors were detected against all these isoforms. Some of them are exemplified in the Table 1 where we can observe low nanomolar and subnanomolar inhibitory activities against the isoforms involved in pathologies such as glaucoma (CA II and XII) or cancer (CA IX).

Table 1. CA I, II, IX, and XII inhibition data with some examples of DTCs from the work by Carta et al.12

			KI (nM)^a
	R1	R2	hCAI	hCAII	hCAIX	hCAXII
1	Ph–	H–	4.8	4.5	4.2	4.3
2	O[(CH2CH2)2]N–	H–	4.8	3.6	29.1	9.2
3	Ph–CH2–	H–	4.1	0.7	19.2	11.5
4	O[(CH2CH2)2]		0.88	0.95	6.2	3.4
5	HO(CH2)2–	HO(CH2)2–	9.2	4	4.3	4.2
6	Iso-Bu–	Iso-Bu–	0.97	0.95	4.5	0.99

^aMeans from three different assays. Errors were within ±5–10% of the reported values (data not shown).

Based on one of the most potent CAIs detected in this study, the authors showed that inhibitor 4 was effective in vivo with IOP lowering effects in an animal model of glaucoma. Being water-soluble, with the pH of the solution in the neutral range and with duration of action lasting up to 4–8 h, this new class of CAIs may constitute interesting candidates for developing novel antiglaucoma therapies12.

Two recent studies13^,14 also describe series of N-mono- and N,N-disubstituted DTC as effective inhibitors of β-Cas not only from the bacterial pathogen Mycobacterium tuberculosis (mtCA 1 (Rv1284) and mtCA 3 (Rv3273)) but also from the fungal pathogens Cryptococcus neoformans, Candida albicans and Candida glabrata. All these enzymes were inhibited with efficacies between the subnanomolar to the micromolar one, depending on the substitution pattern at the nitrogen atom from the dithiocarbamate zinc-binding group. Aryl, arylalkyl-, heterocyclic as well as aliphatic DTCs led to potent fungal and bacterial β-CA inhibitors in both the N-mono- and N,N-disubstituted dithiocarbamate series. These new compounds may have the potential for developing antifungal or antibacterial agents with a diverse mechanism of action compared to the clinically used drugs for which many strains exhibit multi-drug resistance2.

The mechanism of action of DTC inhibitors has been elucidated and structural studies have been reported on three N,N-disubstituted dithiocarbamates (Figure 2) by means of high-resolution X-ray crystallography allowing to elucidate the mechanism of action of this new class of potent CAIs12^,15.

Figure 2. Structure of the three N,N-disubstituted dithiocarbamates.

These DTCs inhibited isoform hCA II with K_Is of 0.95, 25.4 and 40.8 nM, respectively. As illustrated for compound 4 (Figure 3), the binding mode of the dithiocarbamate function was shown to be identical to that of trithiocarbonate (Figure 1), with one sulfur atom coordinated to the zinc ion (Figure 3). Variability of orientation was demonstrated for the organic scaffold of the inhibitor, this latter making extensive contact with several amino acid residues from the active site. It was observed, for example, that the benzyl (hydrophobic) moiety of 7 was orientated toward the hydrophilic half of the hCA II active site. In the case of compound 8 the cyano fragment pointed toward the hydrophilic part whereas the phenyl moiety was orientated toward the hydrophobic half of the cavity. The small and compact morpholine dithiocarbamate (4) was found in the middle of the active site cavity12^,15.

Figure 3. Electronic density for the adduct of dithiocarbamate 4 bound within the active site of human carbonic anhydrase (hCA II). The zinc ion is shown as the central sphere, and the amino acid residues involved in the binding are evidenced and numbered12.

Recently Carta et al. reported new zinc-binding motifs analogues to the DTC: xanthates () and thioxanthates (organic trithiocarbonates, ), which have mechanism of action different from that of the sulfonamides but similar to that of the DTCs16.

A library of derivatives with all types of activities was reported from micromolar to low nanomolar inhibitors against four isoforms: the cytosolic hCA I and II and the transmembrane hCA IX and XII. Furthermore, isoform-selective compounds were identified against all these CAs: 13 was a selective hCA II-inhibitor, 9 and 10 were selective hCA I-inhibitors, whereas xanthates 11 and 12 and the thioxanthate 15 were hCA IX/XII selective (over hCA I and II) inhibitors.

Some of the most effective hCA II inhibitor xanthates were evaluated in vivo, in animal models of glaucoma in normotensive/hypertensive rabbits, and showed effective IOP lowering after direct administration within the eye. The best compound was 14 (K_I of 5.4 nM against hCA II), which led to an IOP lowering of 12 mmHg (3 times as high as the equivalent concentration of the clinically used Dorzolamide), t = 100 min postadministration. This effect was rather prolonged, for more than 250 min.

To understand the CA inhibition mechanism with xanthates and thioxanthates, some theoretical studies were reported showing that the binding mode of these compounds to hCA II closely resembles the binding of the DTCs, which has been investigated by means of X-ray crystallography (Table 2). moiety is directly coordinated to the Zn (II) ion from the enzyme active site and also participates in an interaction (hydrogen bond) with the OH moiety of Thr199, whereas the organic scaffold is deeply buried within the enzyme active site participating in favorable hydrophobically interactions with amino acid residues involved in the binding of other classes of CAIs (such as the sulfonamides and the DTCs), among which are His64, Phe131, and Leu198.

Table 2. CA I, II, IX, and XII Inhibition data with some examples of xanthate and thioxanthate from the work by Carta et al16.

			KI (nM)^a
	R		hCAI	hCAII	hCAIX	hCAXII
9	Boc-NH–(CH2)4–		5.6	9.5	622	705
10	Boc-NH–(CH2)6–		6.3	59	765	744
11	Ph-CH2-		76.1	45.1	8.1	6.0
12	2-Pyridyl-(CH2)2–		74.2	13.1	4.3	3.6
13	–		63.4	6.6	643	835
14	Ph2CH–CH2–		64.1	5.4	58	63.3
15	Ph–CH2–		73.5	75.7	7.8	6.7

^aMeans from three different assays. Errors were within ±5–10% of the reported values (data not shown).

Hydroxamic acid and salicylaldoxime

Hydroxamic acid

Hydroxamic acids have been extensively studied as a pharmacophore group because of their ability to coordinate with metal ions within metalloenzymes such as urease and metallopeptidase. Nevertheless, their interaction with the different α-CA isozymes has been only poorly described. Some studies have been reported concerning CA inhibition studies of related compounds such as sulfonylated amino acyl hydroxamates17, sulfonylated hydroxamates18 and iminodiacetyl-based hydroxamate-benzenesulfonamide conjugates19. Only recently Di Fiore et al. reported investigations related to systematic comparative inhibition studies of a very simple model compound phenyl hydroxamate (16) against the 12 catalytically active hCA isoforms20 (Figures 4 and 5).

Figure 4. (A) Active site region in the hCA II – 16 complex. (B) Zn²⁺ coordination geometry of N-(hydroxy)-benzamide 16²⁰.

Figure 5. Structure of compounds 16 and 17.

It was shown that compound 16 was able to inhibit all 12 CA isoforms, with inhibition constants in the range of 0.94–179 μM, thus being less effective as CAI compared to the benzene sulfonamide.

The X-ray crystal structure of compound 16 bound to hCA II also afforded interesting hints regarding the versatility of the hydroxamate as a ZBG for designing CAIs (Figure 4). Indeed, it was demonstrated that depending on the nature of the R moiety, this ZBG can adopt different coordination modes to the catalytic zinc ion within the CA active site. These findings suggest that the enzyme–inhibitor interaction of this new CAI class can be largely modulated by exploring different substitution patterns at the R group20.

Very recently, a biological application of this new family of CA inhibitors was reported showing that compound 17 may target two proteins that are important for the parasite life cycle: α-carbonic anhydrase (TcCA) and the metalloproteinases21 (Figure 5). This compound was active on the growth inhibition of the three developmental forms of the parasite and showed a selectivity index (SI) of 6.7 with no cytotoxicity to macrophage cells. Preliminary in vivo data showed that 17 was more effective than the standard drug benznidazole being able to reduce bloodstream parasites in all treated mice17.

Salicylaldoxime

The salicylaldoxime scaffold was recently identified as new interesting zinc-binding motif by Tuccinardi et al.22 who reported new effective inhibitors of CAs. A library of eight compounds was described and their inhibitory activity against four CA isoforms (hCA I, II, IX, XII) was evaluated. All the analysed compounds showed inhibition potencies (Ki) against the tested isoforms in the range of 2.3–10.2 μM. Examples of three of the best inhibitors obtained in this study are depicted in Figure 6.

Figure 6. Structures of salicylaldoxime 18–20.

A computational approach was described by the author for compound 18 to analyze the binding mode of these new types of inhibitors with carbonic anhydrases and, in particular, to disclose the binding geometry of the ZBG. It was suggested that the salicylaldoxime moiety binds the zinc ion through the oxime oxygen atom that also forms an H-bond with Thr199. These preliminary results indicate that the salicylaldoxime platform can be considered as a new zinc-binding motif for the development of new carbonic anhydrase inhibitors

Conclusion

The sulfonamides and their isosteres (sulfamates, sulfamides, etc.) are the classical CAIs which act by binding to the metal ion from the active site and replacing the nucleophile (water molecule/hydroxide ion) employed in the catalytic cycle. However, recently new chemotypes possessing this inhibition mechanism have been reported. Based on the X-ray crystal structure of the adduct of the simple inorganic anion trithiocarbonate with the predominant human isoform, CA II, at least three novel classes of CAIs were discovered: the dithiocarbamates, xanthates and thioxanthates. It has been shown that these CAIs potently inhibit enzymes belonging to the α- and β-CA classes. The hydroxamates constitute another class of recently studied CAIs both against mammalian (hCA I, II, IX and XII) and protozoan (T. Cruzii) enzymes. By means of X-ray crystallographic studies it has been demonstrated that the CONHOH is a very versatile zinc-binding function, coordinating to the Zn(II) ion in a variety of modes (depending on the organic scaffold to which this moiety is attached). Another chemotype for which CA inhibitory properties were recently reported is the salicylaldoxime scaffold. X-ray crystal structures were reported for CA II complexed with dithiocarbamates and hydroxamates but not for the xanthates and salicylaldoximes, which were investigated by kinetic measurements and docking studies. The dithiocarbamates and the xanthates also showed potent antiglaucoma activity in animal models of the disease, whereas some hydroxamates inhibited the growth of Trypanosoma cruzii probably by inhibiting the protozoan CA.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.