Sulfocoumarins as dual inhibitors of human carbonic anhydrase isoforms IX/XII and of human thioredoxin reductase

Abstract The hypothesis that sulfocoumarin acting as inhibitors of human carbonic anhydrase (CA, EC 4.2.1.1) cancer-associated isoforms hCA IX and – hCA XII is being able to also inhibit thioredoxin reductase was verified and confirmed. The dual targeting of two cancer cell defence mechanisms, i.e. hypoxia and oxidative stress, may both contribute to the observed antiproliferative profile of these compounds against many cancer cell lines. This unprecedented dual anticancer mechanism may lead to a new approach for designing innovative therapeutic agents.


Introduction
Earlier, we reported 6-substituted sulfocoumarins 1 1 (designed as isosteres of the structurally related coumarins 2-6 ) as potent and remarkably isoform-selective inhibitors of the metallo-enzyme carbonic anhydrase (CA, EC 4.2.1.1) 7,8 . The ability of sulfocoumarins to selectively inhibit membrane-bound hCA IX and XII isoforms were attributed to the unique mechanism of action of these compounds whereby they act as prodrugs activated by CA-mediated hydrolysis [1][2][3][4][5][6] . This makes these inhibitors fundamentally different from the classical carbonic anhydrase inhibitors (CAIs)e.g. those of sulphonamide type which act by binding to the CA prosthetic zinc ion present in all isoforms, which makes designing isoformselective sulphonamide CAIs particularly difficult. On the contrary, CA-mediated hydrolysis of sulfocoumarins 1 (as well as their progenitors coumarins) leads to the in situ formation of the Z-configured stiryl sulphonic acid (Z)-2 which is likely to isomerise to (E)-2, the active inhibitor form whose binding to CA was confirmed by X-ray crystallography 1 . This inhibitor activation and binding apparently occurs only in the protein environment of the two membrane-bound isoforms (hCA IX and XII) which makes these mechanistically distinct inhibitors ideal tools for targeting hypoxia survival mechanism in tumour cells providing which overexpression of precisely these two isoforms is considered responsible for 9 . Indeed, selective targeting of hCA IX and XII has been confirmed to lead to retardation of tumour growth and, ultimately, reduction of tumour size 10 .
Another principal mechanism of tumour survival which we have been recently tackling 11,12 as a target for anticancer agent design, is that providing tumour cell defence against oxidative stress (reactive oxygen species or ROS). In particular, tumour cells have been shown to overexpress thioredoxin reductase (TrxR, EC 1.8.1.9) which contributes to their resistant phenotype characterised by higher levels of ROS 13 . Thus, targeting TrxR1 (the most widespread cytosolic isoform of human TrxR) has been investigated as an emerging approach to selective killing of cancer cells 14 . This selenocysteine (Sec) enzyme, along with NADPH and thioredoxin (Trx) is part of the Trx system and responsible for maintaining Trx in its reduced bis-sulfhydryl state. Among several classes of inhibitors of varying degree of electrophilicity towards the catalytic Sec residue (recently reviewed by Bellelli 15 and Fang 16 ), we found covalent Michael acceptor inhibitors (such as Ugi-type adducts 3 which we dubbed "Ugi Michael Acceptors" or UMAs) to be particularly efficacious 12 . The mechanism of inhibitory action of UMAs towards TrxR1 likely involves the irreversible covalent trapping of the selenide group of the catalytic Sec residue (which exists in the ionised form at physiological pH 17 ) by the electrophilic b-benzoylacrylamide moiety present in 3.
Considering the presence of a potential Michael acceptor moiety in sulfocoumarins 1, we hypothesised that in addition to their inhibitory activity towards hCAs, these compounds could potentially act as Michael acceptor-type TrxR inhibitors (Figure 1), thus acting as dual inhibitors which target two cancer cell defence mechanisms at a time. Herein, we present our preliminary results obtained in the course of verifying this hypothesis.

Chemical synthesesgeneral
Reagents and starting materials were obtained from commercial sources (Sigma-Aldrich, St. Louis, MO) and used as received. The solvents were purified and dried by standard procedures prior to use; petroleum ether of boiling range 40-60 C was used. Flash chromatography was carried out using Merck silica gel (230-400 mesh). Thin-layer chromatography was performed on silica gel, spots were visualised with UV light (254 and 365 nM). Melting points were determined on an OptiMelt automated melting point system. IR spectra were measured on a Shimadzu FTIR IR Prestige-21 spectrometer. NMR spectra were recorded on Varian Mercury (400 MHz) spectrometer with chemical shifts values (d) in ppm relative to TMS using the residual DMSO-d 6 signal as an internal standard. Elemental analyses were performed on a Carlo Erba CHNSeO EA-1108 apparatus. Starting material sulfocoumarins (4 18 and 5 19 ) were prepared as described previously. Alkynes employed in the synthesis of 1a-b are commercially available. Tetrazoles employed in the synthesis of 1c-d were prepared according to the literature protocols 20,21 . All reagents for biological assays were purchased from Sigma (St. Louis, MO).

Carbonic anhydrase inhibition assay
An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalysed CO 2 hydration activity 22 . Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 20 mM Tris (pH 8.3) as buffer, and 20 mM Na 2 SO 4 (for maintaining constant the ionic strength), following the initial rates of the CA-catalysed CO 2 hydration reaction for a period of 10-100 s. The CO 2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor, at least six traces of the initial 5-10% of the reaction have been used for determining the initial velocity. The uncatalysed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled-deionised water and dilutions up to 0.005 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were pre-incubated 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 and the Cheng-Prusoff equation, as reported earlier, and represent the mean from at least three different determinations. All CA isoforms were recombinant ones obtained in-house 23-26 .

TrxR activity by DTNB reduction assay
Determination of TrxR activity in SHSY5Y cell lysate. TrxR activity in cell lysate was measured in 96-well plates using previously described methods 27,28 . For TrxR activity measurement, compounds of different concentrations were incubated with 50 mg of cell lysate and 200 mM NADPH in a volume of 100 mL of 50 mM Tris-HCl and 1 mM EDTA, pH 7.5 (TE buffer), for different time points in 96-well plates at room temperature. Then, 100 mL of TE buffer containing DTNB and NADPH was added (final concentration: 2.5 mM and 200 mM, respectively), and the linear increase in absorbance at 412 nm during the initial 2 min was measured with a Tecan Infinite M1000 multifunctional microplate reader. TrxR activity was calculated as a percentage of enzyme activity of that of DMSO vehicle treated sample.

Cytotoxicity assay
Thus, monolayer tumour cell lines HT-1080 (human fibrosarcoma), SHSY5Y (human neuroblastoma), and MCF-7 (breast adenocarcinoma) were cultured in standard medium DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% foetal bovine serum. About 2000-4000 cells per well (depending on line nature) were placed in 96-well plates and after 24 h compounds were added to the wells. Untreated cells were used as a control. The plates were incubated for 48 h, 37 C, and 5% CO 2 . The number of surviving cells was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolinium bromide (MTT). MTT-test: after incubating culture medium was removed and 200 mL fresh medium with 20 mL MTT (2 mg/mL in HBSS) was added in each well of the plate. After incubation (3 h, 37 C, 5% CO 2 ), the medium with MTT was removed and 200 mL DMSO were added at once to each sample. The samples were tested at 540 nm on Thermo Scientific Multiskan EX microplate photometer. The half-maximal inhibitory concentration (IC 50 ) of each compound was calculated using Graph Pad Prism V R 3.0 (GraphPad Software, La Jolla, CA).

Biological evaluation
To our utmost delight, when the previously established 18,19 potent and selective inhibitory profile of compounds 1a-d towards cancer-related hCA IX and hCA XII isoforms was confirmed in reference to known CAI acetazolamide (AAZ), we have also found these compounds to display dose-dependent inhibition of TrxR activity in SHSY5Y cell lysate with IC 50 values confidently residing in the 10 À5 … 10 À4 M range. Adding to the satisfaction over having our initial hypothesis regarding the dual CA/TrxR inhibitory effects of compounds 1, rather potent antiproliferative activity was established as evaluated against cultures of cancer cells such as HT-1080 (human fibrosarcoma), SHSY5Y (human neuroblastoma), and MCF-7 (breast adenocarcinoma). These findings are summarised in Table 1.

Conclusions
The previously described sulfocoumarins that were shown to potently and selectively inhibit cancer-related hCA IX and hCAXII isoforms (whose overexpression is a well-established mechanism of tumour cell defence against hypoxia) also display noticeable, dose-dependent inhibition of TrxR activity in cancer cell lysates. As overexpression of TrxR in cancer cells is a defence mechanism against oxidative stress, the established dual inhibition pattern constitutes a significant starting point for the design and discovery of new anticancer agents based on the dual targeting of the two defence mechanisms crucial for cancer cell survival. This communication opens a new line of research in our laboratories aimed at investigating the practical aspects of the new dual inhibitor design and establishing a well-understood link between inhibition of these two enzyme groups and the dual inhibitors' antitumor activity. The results of this research will be reported in due course.

Disclosure statement
No potential conflict of interest was reported by the authors.