Abstract
A series of halogenated sulfanilamides and halogenated benzolamide derivatives have been investigated as inhibitors of three β-carbonic anhydrases (CAs, EC 4.2.1.1) from the bacterial pathogen Mycobacterium tuberculosis, mtCA 1 (Rv1284), mtCA 2 (Rv3588c) and mtCA 3 (Rv3273). All three enzymes were inhibited with efficacies between the submicromolar to the micromolar one, depending on the substitution pattern at the sulfanilamide moiety/fragment of the molecule. Best inhibitors were the halogenated benzolamides (KIs in the range of 0.12–0.45 μM) whereas the halogenated sulfanilamides were slightly less inhibitory (KIs in the range of 0.41–4.74 μM). This class of β-CA inhibitors may have the potential for developing antimycobacterial agents with a diverse mechanism of action compared to the clinically used drugs for which many strains exhibit multi-drug/extensive multi-drug resistance.
Introduction
The genome of the human pathogen Mycobacterium tuberculosis contains at least three β-carbonic anhydrases (CAs, EC 4.2.1.1), called mtCA 1, 2 and 3, and encoded by the genes Rv1284, Rv3588c Rv3273Citation1–6. These enzymes have been cloned and their catalytic activity and inhibition profiles with sulfonamides recently investigatedCitation3–6. As most CAs described to date, these enzymes are inhibited by sulfonamides and related compoundsCitation8–12. Indeed, interesting, low nanomolar or subnanomolar sulfonamide inhibitors targeting these CAs have already been detectedCitation3–6; it is not yet clear whether the in vivo inhibition of these enzymes has an antimycobacterial effectCitation7,Citation12.
Resistance to antibiotics belonging to several different classes is escalating and represents a worldwide problemCitation13–17. Many strains of Gram-negative/positive bacteria (such as among others Staphylococcus aureus, M. tuberculosis, Helycobacter pylori, Brucella suis, Streptococcus pneumoniae, etc.) no longer respond to various classes of antibioticsCitation13–17. Cloning of the genomes of bacterial pathogens offers however the possibility to explore alternative pathways for inhibiting virulence factors or proteins essential for the pathogens life cycle. Among the many new such proteins recently explored, CAs emerged as interesting drug targetsCitation12,Citation18. Indeed, potent sulfonamide inhibitors of the bacterial CAs from H. pylori, B. suis, S. pneumoniae and several other such pathogens were shown to inhibit the growth of the pathogen, by a mechanism of action not entirely understood at this momentCitation12,Citation18–22.
The classical CA inhibitors (CAIs) are the primary sulfonamides, RSO2NH2, which are used clinical for more than 50 years as diuretics or systemically acting antiglaucoma drugsCitation8,Citation9,Citation23,Citation24. Around 30 clinically used drugs (or agents in clinical development) belonging to the sulfonamide or sulfamate class, show significant CA inhibitory activity and are used for the management of a variety of disordersCitation8,Citation9,Citation23. However, all these drugs target all mammalian CAs, of which 16 different isoforms are known so far, and as thus, they may show undesired side effectsCitation8,Citation9,Citation23. Ultimately, a large number of such derivatives started to be investigated as anti-infectives, which target bacterial/fungal CAs (generally belonging to the β-CA class), but this field is still a very new one, with non-irrelevant resistance from the scientific community in accepting these enzymes as anti-infective targetsCitation8,Citation12.
Bacteria predominantly encode for β-class CAs, which are not present in vertebrates, and these enzymes were started to be seriously considered as possible drug targets for obtaining antibacterials devoid of the resistance problems mentioned above, which affect most classes of antibiotics in clinical useCitation13–16.
Thus, exploring various chemotypes among the sulfonamides may lead to the discovery of β-CA inhibitors with good affinity for enzymes from pathogenic species. Here, we report that the sulfonamides incorporating halogenated benzolamide/sulfanilamide moieties, a class of α-CAIs reported by our groupCitation25, also act as highly effective inhibitors of the three β-CAs from M. tuberculosis, i.e. mtCA 1–mtCA 3.
Materials and methods
Chemistry
Enzymology
mtCA 1–mtCA 3 were recombinant enzymes obtained as described earlierCitation1–7.
CA catalytic activity and inhibition assay
An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalysed CO2 hydration activityCitation26. 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, for α-CAs) or TRIS (pH 8.3 for β-CAs) as buffers, and 20 mM Na2SO4 (for α-CAs) or 20 mM NaCl− for β-CAs (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, 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 inhibitor (10 mM) were prepared in distilled-deionized water and dilutions up to 0.01 nM 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-square methods using PRISM 3, whereas the kinetic parameters for the uninhibited enzymes from Lineweaver-Burk plots, as reported earlierCitation3–7,Citation27,Citation28, and represent the mean from at least three different determinations.
Results and discussions
We report here the investigation of a series of sulfonamides of type 1–10 for their interaction with three β-CAs from the bacterial pathogen M. tuberculosis, i.e. mtCA 1 (Rv1284), mtCA 2 (Rv3588c) and mtCA 3 (Rv3273). The rationale for investigating these compounds as inhibitors of these enzymes is based on the fact that benzolamide BZA, an orphan drug belonging to the CAIsCitation8, is one of the best (submicromolar) sulfonamide inhibitor of the mycobacterial CAs, as reported earlier by our groupCitation3–7.
The following structure-activity relationship (SAR) can be observed from data of :
Table 1. Inhibition data of mycobacterial β-CA isoforms mtCA 1–3 with sulfonamides 1–10 and benzolamide BZA by a stopped-flow, CO2 hydrase assayCitation25.
| (i) | Against mtCA 1 the sulfonamides 1–10 behaved as efficient inhibitors, with inhibition constants in the range of 0.12–1.27 μM, comparable (but slightly better in many cases) to that of the lead molecule sulfonamide, BZA which has a KI of 0.81μM (). A quite straightforward SAR may be observed for the inhibition of this enzyme with the sulfonamides 1–10 investigated here. Thus, the best mtCA 1 inhibitors were the halogenated benzolamides 1–3, for which efficiency increased with the atomic weight of the halogen atom present in the molecule. Indeed, the iodo-fluoro-substituted compound 3 was the best inhibitor detected here (a 6.75-fold increase of activity compared to BZA), followed by the bromo-fluoro-substituted benzolamide 2 and the monochloro-derivative 1. For the halogenated sulfanilamides 4–10, activity was also good, with the best inhibitors being the bromo-chloro-derivative 8 and the dibromosulfanilamide 10 (KIs of 0.41–0.58 μM). It may be observed that the nature of the halogen atoms substituting the sulfanilamide ring or the benzenesulfonamide fragment of the molecule (in the BZA derivatives), is the main factor influencing activity. | ||||
| (ii) | mtCA 2 was also inhibited efficiently by sulfonamides 1–10, with inhibition constants in the range of 0.41–4.74 μM. As BZA, its halogenated derivatives 1–3 were the best mtCA 2 inhibitors, with KIs in the range of 0.41–0.46 μM. Thus, for this enzyme, the presence of the halogeno substituents in the BZA scaffold does not significantly increase potency as mtCA 2 inhibitors (whereas, as discussed above, for mtCA 1 a significant such increase has been observed). The halogenated sulfanilamides 4–10 were roughly an order of magnitude less inhibitory compared to the benzolamides 1–3 and BZA, with KIs in the range of 4.14–4.74 μM. Thus, a very flat SAR is observed in this case. | ||||
| (iii) | The third bacterial CA investigated here, mtCA 3, was also sensitive to inhibition with sulfonamides 1–10, these derivatives show KIs in the range of 0.17–3.19 μM. The lead molecule BZA was also an effective mtCA 3 inhibitor, with KI of 0.34 μM (). Again the best inhibitors detected in this study were the benzolamides. Substitution of one or two hydrogen atoms from the lead BZA with halogens led to a slight increase (2-fold) of the inhibitory power, the most effective compound being the monochloro-derivative 1 (KI of 0.17 μM). The presence of additional halogens or the increase of their atomic weight, as in 2 and 3, leads to a slight decrease of potency but the compounds remain active, similar to the lead BZA. The halogenated sulfanilamides were again around one order of magnitude less inhibitory compared to the benzolamides, with KIs in the range of 2.03–3.19 μM (). The best substitution pattern seems to be the one with two bromine atoms (compound 10) which with a KI of 2.03 μM is the most effective in this series. | ||||
| (iv) | mtCA 1 was the most prone to be inhibited by sulfonamides 1–10, followed by mtCA 3 whereas mtCA 2 was the least inhibited isoform. This is quite different from the inhibition profile of the lead BZA. Indeed, for this compound, the best inhibition was seen against mtCA 3, followed by mtCA 2 and the least inhibited isoform was mtCA 1. | ||||
One of the main problems with mtCA inhibitors investigated so far is that no inhibition of the bacterial growth in vivo has been observed to date (unpublished results from this laboratory and ref.Citation21), probably due to the very hydrophilic character of sulfonamides and their difficulty to cross the thick mycobacteria cell wallCitation21. The rationale of this study was just to increase the lipophilicity of the sulfonamide inhibitors which may allow a better penetrability profile to such compounds. Further studies are thus warranted to obtain highly effective and lipophilic mtCA inhibitors and prove their efficacy in vivo.
Conclusion
We evaluated a series of sulfonamide as inhibitors of three β-CAs from the bacterial pathogen M. tuberculosis, mtCA 1 (Rv1284), mtCA 2(Rv3588c) and mtCA 3 (Rv3273). These enzymes were inhibited with efficacies between the submicromolar to the micromolar one, depending on the substitution pattern at the sulfanilamide moiety/fragment of the molecule. Best inhibitors were the halogenated benzolamides (KIs in the range of 0.12–0.45 μM) whereas the halogenated sulfanilamides were slightly less inhibitory (KIs in the range of 0.41–4.74 μM) against all isoforms. This class of β-CA inhibitors may have the potential for developing antimycobacterial agents with a diverse mechanism of action compared to the clinically used drugs for which many strains exhibit multi-drug/extensive multi-drug resistance.
Declaration of interest
This work was supported by an EU FP7 research grant (Metoxia project).
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