Appraisal of anti-protozoan activity of nitroaromatic benzenesulfonamides inhibiting carbonic anhydrases from Trypanosoma cruzi and Leishmania donovani

Abstract Chagas disease and leishmaniasis are neglected tropical disorders caused by the protozoans Trypanosoma cruzi and Leishmania spp. Carbonic anhydrases (CAs, EC 4.2.1.1) from these protozoans (α-TcCA and β-LdcCA) have been validated as promising targets for chemotherapic interventions. Many anti-protozoan agents, such as nitroimidazoles, nifurtimox, and benznidazole possess a nitro aromatic group in their structure which is crucial for their activity. As a continuation of our previous work on N-nitrosulfonamides as anti-protozoan agents, we investigated benzenesulfonamides bearing a nitro aromatic moiety against TcCA and LdcCA, observing selective inhibitions over human off-target CAs. Selected derivatives were assessed in vitro in different developmental stages of T. cruzi and Leishmania spp. A lack of significant growth inhibition has been found, which has been connected to the low permeability of this class of derivatives through cell membranes. Further strategies necessarily need to be designed for targeting Chagas disease and leishmaniasis with nitro-containing CA inhibitors.


Introduction
Chagas disease (American trypanosomiasis) and leishmaniasis are potentially life-threatening illnesses that have been included in the list of neglected tropical diseases (NTDs) by the World Health Organization (WHO). These infections belong to the vector-borne diseases affecting 20 million people and killing more than 50,000 every year and are caused by parasites of the kinetoplastida family (Trypanosoma cruzi and Leishmania sp.) 1 . Kissing bugs of the Triatoma and Rhodnius genera naturally transmit T. cruzi that is primarily diffused in Latin America. Chagas disease progresses by damaging organs in the cardiac, digestive, or nervous systems 1 . The bite of infected phlebotomines instead is the main cause of Leishmania transmission and potentially generates skin or visceral fatal damages. Leishmaniasis is the first-in-class NTD in terms of mortality and morbidity 1 .
To date, a limited arsenal of anti-protozoan agents is available for the treatment of these NTDs. These drugs are marked by high toxicity and limited efficacy, and resistance phenomena are constantly increasing worldwide [2][3][4] . The poor interest shown by the pharmaceutical industry in searching new effective drugs for NTDs treatment is related to high costs and expected low financial return. On the contrary, it should be considered a priority to find new approaches in the treatment of these parasitosis 2,5 . Large-scale analysis on the completely known genome sequence of both protozoans have recently provided the identification of new enzymatic targets 6,7 .
The enzymes carbonic anhydrases (CAs, EC 4.2.1.1) identified in these protozoans, TcCA in T. cruzi and LdcCA in L. donovani (a parasite from the Leishmania complex, causing visceral leishmaniasis) have recently been recognised as suitable targets to fight these infections 6,8,9 . CAs are natural catalysts that speed up the rate of CO 2 conversion to bicarbonate and proton. This reaction was shown to be basic in the growth and virulence of pathogenic microorganisms 9 . TcCA and LdcCA were both cloned and characterised in 2013 [10][11][12] . Many inhibitors of these isoforms have been identified, which represent potential anti-protozoan agents acting by a new mechanism of action which is probably devoid of crossresistance to the existing drugs.
LdcCA is a b-class CA whose catalytic activity evaluation reported a k cat of 9.35 Â 10 5 s -1 , K m of 15.8 Â 10 À3 M, and k cat /K m of 5.9 Â 10 7 M -1 s -1 12 . LdcCA was shown to be efficiently inhibited by sulfonamides, heterocyclic thiols, and N-nitrosulfonamides with nanomolar inhibition constants 12,18 . Some compounds of the two latter classes showed in vitro anti-leishmanial activity in preliminary assays, causing the reduction of the parasites growth and their death 12,18 .
N-Nitrosulfonamides have been designed by us based on the presence of the nitro group in the structure of many anti-protozoan agents, such as the nitroimidazoles, this moiety being pivotal for the drug mechanism of action 18,19 . For instance, nifurtimox and benznidazole (Bzn) have been the first effective drugs for treating acute-phase human Chagas infection, with the first being no longer available on the market because of undesirable side effects 6 . Considering that sulfonamides are the most effective CAIs known to date 20 , we first attached the nitro group on the sulfonamide itself, providing the N-nitro derivatives as a new chemotype exhibiting a selective inhibition of protozoan CAs over human ubiquitous isoforms 18 . As second design strategy, we report herein, consists in the incorporation of the nitro group on the benzene scaffold bearing the sulfonamide, driven by the aromatic character shown by the nitro moieties present in many anti-protozoan agents, mentioned above. A set of 3-nitrobenzenesulfonamide bearing a variety of substituents on the main scaffold has thus been reported. This set has been recently evaluated also for the inhibition of the human tumour-associated CA IX and XII over the ubiquitous CA I and II and for hypoxia-enhanced anti-proliferative activity on tumour cell lines 21 . In fact, nitroaromatic groups are subjected to bioreduction processes in hypoxic tissues, which can be exploited to selectively generate cytotoxins against tumour cells 21 . Here, the set of nitro-benzenesulfonamides has been screened for the inhibition of TcCA and LdcCA and the most effective derivatives were studied in vitro against different species of Leishmania and T. cruzi.

Carbonic anhydrase inhibition
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 Hepes (pH 7.5) 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 0.01 nM were done thereafter with the assay buffer. 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 nonlinear least-squares methods using PRISM 3 and the Cheng-Prusoff equation, as reported earlier, and represent the mean from at least three different determinations 23-26 . All CA isoforms were recombinant ones obtained in-house as reported earlier 27,28 .

Inhibitory activity on epimastigotes of Trypanosoma cruzi and promastigotes of Leishmania
The evaluation of anti-parasites activity was performed in 96 well plates where the synthetic compounds were serially diluted in the PHBIL medium supplemented with 10% FBS in concentrations ranging from 2 to 400 mM. Then, parasites (1.8 Â 10 6 ) were added to each well and the plates incubated for 48 h at 28 C. The experiment controls were: negative control (culture medium without parasite) and positive culture (culture medium with parasite). Benznidazole and amphotericin B (Amp) were used as reference drugs of T. cruzi and Leishmania, respectively. The minimum inhibitory concentration (MIC) for epimastigotes (T. cruzi DM28c and Y) and promastigotes (L. amazonensis and L. infantum) was performed using resazurin (125 mM) as an indicator of cellular metabolic function. MIC was determined as the lowest concentration of the inhibitor capable of inhibiting in vitro growth of the parasites by spectrophotometric analysis at 490 and 595 31 . The concentration of drug which reduces parasites number by 50% (IC 50 ) was determined by regression analysis using Microsoft Excel 2013.

Cytotoxicity assay in macrophages
Cytotoxicity was performed using tetrazolium dye (MTT) colorimetric assay. RAW 264.7 macrophages cells were harvested after confluent monolayer achievement 32 . The cells were washed twice with PBS and a cellular suspension of 10 6 cells/ml was prepared in fresh DMEM culture medium. Aliquots of 100 ml of the cellular suspension were placed into polystyrene 96-well plates, and then incubated at 37 C in a 5% CO 2 atmosphere for 6 h in order to allow macrophage adherence. After this period, the adherent cells were subjected to treatment with several concentrations of the drugs (2-256 mM), and then incubated for additional 48 h. Finally, 20 ml of MTT solution (5 mg/ml) were added to each well and the plates incubated for 4 h. Macrophage viability was determined after formazan crystals solubilisation with DMSO followed by the absorbance measurement at 570 nm using a SpectraMax M5 spectrophotometer (Molecular Devices, Sunnyvale, CA).

Determination of selectivity index
The selectivity index (SI) of tested drugs was calculated as a ratio of RAW 264.7 macrophages CC 50 to parasites IC 50 . Benznidazole (Sigma-Aldrich, Milan, Italy) and Amp were used as reference drugs.

Carbonic anhydrase inhibition
The TcCA and LdcCA inhibitory profiles of compounds 4-25 were evaluated by applying a stopped flow carbon dioxide hydrase assay 22 in comparison to AAZ as standard CAI and compared to those against the human off-target CA I and II. The following SAR can be built from the inhibition data shown in Table 1.
TcCA was effectively inhibited by most 3-nitrobenzenesulfonamides investigated here. Inhibition constants (K I s) span in medium nanomolar to low micromolar range between 0.08 and 10. LdcCA inhibition profiles show analogies with those against TcCA. Again, the simplest derivatives 4, 6, 8, and 12 act as the best LdcCA inhibitors with K I s of 0.21, 0.34, 0.46, and 0.39 mM, respectively. Benzoylation of the hydroxy moiety at position 4 markedly reduced the LdcCA inhibitory properties of 7, 9, and 10 (K I s of 4.68, 3.87, and 8.49 mM) as well as incorporation of the charged pyridinium portion as in 11 (K I of 6.57 mM). Ureido derivatives 13-25 inhibited LdcCA in a rather flat range spanning from 0.86 to 3.65 mM.
As a general trend, most compounds were more effective against TcCA than CA I, with an SI from 2 to >150, with the exception of 9 and 10 ( Table 2). On the other hand, only few derivatives (8,12,13,18,19,22, and 23) inhibited TcCA more efficiently than CA II, with SI spanning between 2.5 and 6. LdcCA was found to be better inhibited than hCA I by most compound, though the SIs were lower than those TcCA/CA I, and spanned in the range of 2-50. Most compounds inhibited CA II better than LdcCA. All compounds inhibited the screened isoforms worse than the standard AAZ, but the latter did not show selectivity for the target TcCA and LdcCA compared to the ubiquitous hCAs ( Table 2).

Anti-protozoan activity
3.3.1. Trypanosoma cruzi strain DM28c and Y Ten selected derivatives bearing different substituents at the 3nitrobenzenesulfonamide scaffold (4, 6, 8, 10, 11, 17, 18, 19, 21, and 24) were screened for their inhibition activity different species of Leishmania and Trypanosoma cruzi.  The MIC and IC 50 values against T. cruzi epimastigote forms of these compounds are shown in Table 3. The experiments showed that no compounds significantly affect the growth of the pathogen below 256 mM. The reference drug Bzn showed IC 50 values against Dm28c clone and Y strains of 16.56 ± 1.51 and 6.54 ± 1.82 mM, respectively. The assessment of the toxicity of the selected 3-nitrobenzensulfonamides for Raw 267.4 macrophages cells showed that most derivatives were less toxic than Bzn (CC 50 of 115.14 ± 9.48 mM) with CC 50 above 172.65 ± 10.44 mM. Compounds 4 and 6 showed instead comparable toxicity with Bnz with CC 50 values of 97.65 ± 11.13 and 100.21 ± 17.27 mM. macrophages cells compared to Amp, that has a CC 50 of 1 mM against both strains.
Unfortunately, the tested 3-nitrobenzenesulfonamides turned out to be ineffective in vitro against strains of T. cruzi and Leishmania. The lack of activity is not a totally new issue in the field of sulfonamide CAIs against pathogens. For instance, some sulfonamide derivatives demonstrated remarkable in vitro efficacy in inhibiting the b-CA from the yeast Malassezia globosa, arousing anyhow complications in vivo because of permeability problems through biological membranes 33 .
In the context of T. cruzi and Leishmania, some previously tested sulfonamides showed an absence of anti-protozoan efficacy, which has been related to the lack of permeability through the biological membranes of the pathogen [34][35][36] . Hence, a formulation of such sulfonamides in nano-emulsions (NEs) of clove oil was attempted to enhance their bioavailability and penetrability through membranes 34,35 . The drugs-NEs formulations potently inhibited the growth of T. cruzi and Leishmania in vitro, with a huge increase of efficacy over the sulfonamide CAI alone. NEs turned out as a novel vehicle for the delivery of such hydrophilic drugs.
Indeed, it should be noted that 3-nitro-4-hydroxybenzenesulfonamides reported here are even more hydrophilic, which can cause difficulties for the compounds to cross the protozoa cell membrane and inhibit the cytoplasmatic CAs or exert further actions due to the nitro group. Hence, formulation to enhance the compounds bioavailability, such as NEs, is being prepared to evaluate the real anti-protozoan efficacy of these set of nitroaromatic CAIs.

Conclusions
We proposed here nitroaromatic sulfonamides for the treatment of Chagas disease and leishmaniasis based on CA inhibition. As a continuation of a previous work of us on N-nitrosulfonamides as anti-protozoan agents, we studied here benzenesulfonamides (4-24) bearing a nitro moiety on the aromatic scaffold against TcCA from T. cruzi, responsible of Chagas disease, and LdcCA from Leishmania spp. The compounds reported valuable micromolar inhibition of these two enzymes, in some cases even selective for the target CAs over the human ubiquitous CA I and II. Unfortunately, a selected set of such derivatives tested in vitro against multiple strains of T. cruzi and Leishmania did not produce growth inhibition of the parasites. The lack of anti-protozoan efficacy of sulfonamide type derivatives had been already reported by us and justified by low permeability of this class of derivatives through the cell membranes. The use of carriers such as nanoemulsions allowed to overcome this issue. The application of this approach has been being carried out for 3-nitrosulfonamides 4-24 to elucidate whether the combination of CA inhibition and further anti-protozoan actions related to the nitro group could be a winning anti-infective strategy.

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