Mono- and di-thiocarbamate inhibition studies of the δ-carbonic anhydrase TweCAδ from the marine diatom Thalassiosira weissflogii

Abstract The inhibition of the δ-class carbonic anhydrase (CAs, EC 4.2.1.1) from the diatom Thalassiosira weissflogii, TweCAδ, was investigated using a panel of 36 mono- and di-thiocarbamates chemotypes that have recently been shown to inhibit mammalian and pathogenic CAs belonging to the α- and β-classes. TweCAδ was not significantly inhibited by most of such compounds (KI values above 20 µM). However, some aliphatic, heterocyclic, and aromatic mono and di-thiocarbamates inhibited TweCAδ in the low micromolar range. For some compounds incorporating the piperazine ring, TweCAδ was effectively inhibited (KIs from 129 to 791 nM). The most effective inhibitors identified in this study were 3,4-dimethoxyphenyl-ethyl-mono-thiocarbamate (KI of 67.7 nM) and the R-enantiomer of the nipecotic acid di-thiocarbamate (KI of 93.6 nM). Given that the activity and inhibition of this class of enzyme have received limited attention until now, this study provides new molecular probes and information for investigating the role of δ-CAs in the carbon fixation processes in diatoms, which are responsible for significant amounts of CO2 taken from the atmosphere by these marine organisms.


Introduction
The di-thiocarbamates (DTCs) possessing the general formula RR 1 NCS 2 M (where R, R 1 may be H, alkyl, cycloalkyl, aryl, hetaryl, etc., and M is a cation) were recently reported as a new class of inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) 1 . Their inhibitory activity was investigated against aand b-class CAs from various organisms 1,2 and they also led to the discovery of two new CA inhibitor (CAI) classes, the xanthates 3 and the mono-thiocarbamates (MTCs) 4 . Representatives of MTCs and DTCs acting as CAIs are shown in Figures 1 and 2.
Inhibition of CAs belonging to some of the seven genetically distinct families known to date 5-10 has various biomedical applications owing to the fact that these enzymes catalyse a simple but physiologically crucial reaction: the hydration of CO 2 to bicarbonate and hydronium ions [5][6][7][8][9][10] . Interference with this process has important physiological and pathological consequences because CAs are involved in pH regulation, biosynthetic processes, metabolism, secretion of electrolytes, transport of CO 2 /bicarbonate, etc. 5-10 . Their dysregulated expression or activity leads to various pathologies, and as a consequence, their inhibitors are clinically used as diuretics, antiglaucoma, antiepileptic, anti obesity, and antitumour agents [5][6][7][8][9] . Recently, the CAIs were also shown to be effective for the control of neuropathic pain, cerebral ischemia, and some forms of arthritis 10 . The primary sulphonamides and their isosteres (sulphamides and sulphamates) are the main class of CAIs, but in many cases, they indiscriminately inhibit most of the many CA isoforms known in an organism (e.g. 15 CA isoforms belonging to the a-class are known in humans 5,[11][12][13][14][15][16] ). This is the reason why alternative chemotypes, such as the DTCs and MTCs have recently been explored 1-4 . However, this class of CAIs has only been investigated to date for their interaction with human (h), a-class enzymes, and with several CAs from pathogens or model organisms, belonging to the aand b-CA classes 1-4 . The d-CAs were discovered in the diatom Thalassiosira weissflogii 6d , but orthologues of this enzyme have been identified in most diatoms from natural phytoplankton assemblages and are responsible (along with other CAs) for CO 2 fixation by marine organisms 17 . A related species of this diatom, Thalassiosira pseudonana, was shown to possess genes for three a-, five c-, four d-, and one f-CAs 18 . However, none of these enzymes have been cloned and characterised in detail to date, except TweCAd 11 . Diatoms can be considered to be the organisms with the most intricate and poorly understood distribution of CAs, but the roles of these enzymes seem to be crucial for CO 2 fixation and photosynthesis in many organisms and are estimated to be responsible for at least 25% of the inorganic carbon fixation in the oceans 6,17,18 . However, few studies are available for the interaction of d-CAs with modulators of activity, inhibitors, and activators. TweCAd was the only representative of the d-class for which anion and sulphonamide inhibition studies have been reported to date 6d,11 . Here we report the first CA inhibition study with MTCs and DTCs of a d-CA class enzyme, TweCAd, which was cloned and characterised from the marine diatom T. weissflogii 6d,6e .

Materials
MTCs 1-15 4 and DTCs 16-36 1,2 were reported earlier by our group. Reagents/buffers of the highest available purity were obtained from Sigma-Aldrich, Milan, Italy. TweCAd was a recombinant protein produced as reported earlier by our group 6e,11 .

CA enzyme inhibition assay
An Sx.18Mv-R Applied Photophysics (Oxford, UK) stopped-flow instrument has been used to assay the catalytic activity of various CA isozymes for CO 2 hydration reaction 12 . Phenol red (at a concentration of 0.2 mM) was used as indicator, working at the absorbance maximum of 557 nm, with 10 mM Hepes (pH 7.5) as buffer, and 0.1 M Na 2 SO 4 (for maintaining constant ionic strength, which is not inhibitory against TweCAd 11 ), following the CA-catalysed CO 2 hydration reaction for a period of 10 s at 25 C. The CO 2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and activation constants. For each inhibitor at least six traces of the initial 5-10% of the reaction have been used for determining the initial rate. The uncatalysed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitors (10 mM) were prepared in distilled-deionised diluted to 1 nM using the assay buffer. Inhibitor and enzyme solutions were pre-incubated together for 15 min (standard assay at room temperature) prior to assay, in order to allow for the formation of the enzyme inhibitor complex. The inhibition constant (K I ), was obtained by considering the classical Michaelis-Menten equation and the Cheng-Prusoff algorithm by using non-linear least squares fitting as reported earlier 13-16 .

Results and discussion
TweCAd is the only CA belonging to the d-class for which anion and sulphonamide inhibition studies were reported so far 6d,11 . Here, we investigated the inhibition of this enzyme with the panel of MTCs and DTCs of the types 1-36 shown in Figures 1 and 2. The results are shown in Table 1, where for comparison reasons, the inhibition of the human dominant isoforms hCA I and II with the same compounds are reported 1,2,4 . The following structure-activity relationship (SAR) can be obtained from the data of Table 1: (i) A number of MTCs, including 4-6, 10 and the DTCs 20, 21, 23-25, 32, and 33, did not inhibit TweCAd up to 20 mM, although many of these compounds were rather effective inhibitors of hCA I and/or hCA II (Table 1). Such MTCs/DTCs inhibitors are classified as aliphatic, heterocyclic, aromatic, or polycyclic types. Given the structural diversity of such compounds and high inhibition constants, it is challenging to delineate the SAR.
(iii) The MTC/DTCs 1, 2, 7-9, 28, 30, and 34-36 were relatively effective inhibitors of TweCAd, with inhibition constants in the range of 129-997 nM ( Table 1). Some of the MTC and DTCs incorporate the piperazine ring (7-9, 34). In addition, MTC 9 and DTC 34 have the same scaffold but a different ZBG. In this particular case, MTC 9 inhibited TweCAd 6.1-times more efficiently than DTC 34. Interestingly, for the b-CAs, the MTCs were usually much weaker inhibitors compared to the structurally similar DTCs 4 . In addition, the sulphonamide-containing DTC 36 (which contains two potential ZBGs, the sulphonamide and the DTC), there are no net differences of TweCAd inhibitory activity compared to the structurally similar derivatives (e.g. 35) which probably is due to the fact that the DTC in 36 is primarily binding to the metal ion in the enzyme active site, and not the sulphonamide moiety. However, the heterocyclic sulphonamide acetazolamide (AAZ, 5-acetamido-1,3,4-thiadiazole-2-sulphonamide), a clinically used drug 5 , is a much more potent inhibitor (K I of 83 nM) of TweCAd compared to 36 (Table 1).
(iv) The most effective TweCAd inhibitors identified in this MTC/DTC panel were the MTC 12 (K I of 67.7 nM) and the DTC 27 (K I of 93.6 nM). These compounds incorporate scaffolds rather similar to those present in other investigated compounds, which were, however, much less effective as inhibitors of this enzyme. For example, 12 has two methoxy moieties on the scaffold of 11, but there is a difference of activity of 14.7-fold between the two MTCs. The R-enantiomer 27 was on the other hand 5.9 times a more effective inhibitor compared to the S-enantiomer 28. All these data show that small changes in the structure or the stereochemistry of a DTC/MTC lead too dramatic changes of affinity for the target enzyme.
(v) With a few exceptions, TweCAd was less sensitive to this class of CAIs compared to the a-CAs hCA I and II (Table 1). There are several X-ray crystal structures that demonstrate that the DTCs (and presumably also the MTCs) bind to the metal ion in the CA active site by substituting the hydroxide nucleophile that is responsible for the catalytic activity of the enzyme 1,2 . Most probably, this is also the inhibition mechanism by which DTCs and MTCs interact with d-CAs. However, this enzyme class is the least studied of the 7 CA genetic families, and there are no X-ray crystal structures or even homology models available for any d-CAs.
We try to rationalise the obtained inhibition data based on the amino acid sequence of TweCAd, which has been aligned with that of a-CAs for which the X-ray crystal structure is known, of bacterial (HpylCA, a-CA from Helicobacter pylori, SspCA, a-CA from Sulfurihydrogenibium yellowstonensis) or human origin (hCA I and II) (Figure 3). Data of Figure 3 show that for the a-CAs, the zinc Table 1. TweCAd, hCA I, and hCA II Inhibition Data with MTCs 1-15, DTCs 16-36, and acetazolamide (AAZ, 5-acetamido-1,3,4-thiadiazole-2-sulphonamide) as standard drug, by a stopped-flow CO 2 hydrase assay. ligands are three His residues (His94, 96, and119, hCA I numbering system), which align well for the bacterial and human enzymes, whereas the putative zinc ligands of TweCAd do not align at all with those of the a-class enzyme. The same is true for other amino acid residues from the a-CAs, such as the proton shuttle (His64) which is an Asp residue in TweCAd, or residues 106 (a conserved Asp residue in all a-CAs), which is a Thr in TweCAd. Based on these data it is obvious that it is not possible to rationalise the observed SAR with mono-and di-thiocarbamates based only on the sequence of the enzyme, without a homology model or better, an X-ray crystal structure of the diatom enzyme.

Conclusions
The first inhibition study of a d-CA with mono-and di-thiocarbamates, classes of CAIs recently discovered, was reported. TweCAd from the marine diatom T. weissflogii was not particularly sensitive to inhibition by these classes of compounds. Many of the monoand di-thiocarbamates did not show inhibitory action up to 20 mM, whereas some aliphatic, heterocyclic, and aromatic inhibited this enzyme in the low micromolar range. Several MTCs/DTCs incorporating the piperazine ring effectively inhibited TweCAd with K I s in the range of 129-791 nM. The most effective inhibitors identified were 3,4-dimethoxyphenyl-ethyl-mono-thiocarbamate (K I of 67.7 nM) and the R-enantiomer of the nipecotic acid DTC (K I of 93.6 nM). Such inhibitors can now be used as molecular probes to investigate the role of this enzyme in the carbon fixation processes in diatom marine organisms that are responsible for removing large amounts of CO 2 from the atmosphere.

Disclosure statement
The authors do not declare any conflict of interest.   . Multialignment of the TweCAd amino acid sequence with those of bacterial (HpylCA, a-CA from Helicobacter pylori, SspCA, a-CA from Sulfurihydrogenibium yellowstonensis) and human (hCA I and II) a-class enzymes. The zinc ligands of the a-CAs and the putative zinc ligands of TweCAd are evidenced in red, whereas amino acid residues involved in the catalytic inhibition/mechanism (e.g. His64 and Asp106, hCA I numbering) are shown in green and blue, respectively.