https://orcid.org/0000-0003-3342-702X
Abstract
Coumarins were discovered to act as inhibitors of α-carbonic anhydrases (CAs, EC 4.2.1.1) after undergoing hydrolysis mediated by the esterase activity of the enzyme to the corresponding 2-hydroxycinnamic acids. Other classes of CAs among the eight currently known do not possess esterase activity or this activity was poorly investigated. Hence, we decided to look at the potential of coumarins as inhibitors of the η-CA from the malaria-producing protozoan Plasmodium falciparum, PfaCA. A panel of simple coumarins incorporating hydroxyl, amino, ketone or carboxylic acid ester moieties in various positions of the ring system acted as low to medium micromolar PfaCA inhibitors, whereas their affinities for the cytosolic off-target human isoforms hCA I and II were in a much higher range. Thus, we confirm that η-CAs possess esterase activity and that coumarins effectively inhibit this enzyme. Elaboration of the simple coumarin scaffolds investigated here may probably lead to more effective PfaCA inhibitors.
1. Introduction
Comarins were discovered relatively recently to act as inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1)Citation1. Unlike all other inhibitor classes investigated at that time, surprisingly, these compounds were shown to not coordinate to the metal ion from the α-CA active sites (the human isoforms hCA I – XIV were initially investigated for their interaction with these compoundsCitation1,Citation2) but to bind at the entrance of the active site cavity. In addition, the coumarin lactone ring was found hydrolysed to the corresponding 2-hydroxycinnamic acids (either in cis- or trans geometry) making these compounds the first reported class of pro-drug CA inhibitors (CAIs). Thus, a rather large number of drug design studies were performed over the last decadeCitation2–4 using both natural products as well as synthetic coumarins as starting point, which established the fact that coumarjns are among the most effective and isoform-selective CAIs known to dateCitation1–4. Indeed, derivatives with selectivity for all human isoforms have been reported so far, although the largest number of studies and derivatives investigated to date were designed for targeting the transmembrane, cancer-associated isoforms hCA IX and XII, which are validated antitumor/antimetastatic drug targetsCitation5,Citation6.
However, up until now, coumarins were not investigated for their interactions with non-α-CAs. In fact, among the eight reported CA genetic families (the α – ι-CA classesCitation7,Citation8) known so far, only the α-CAs were investigated in detail for their catalytic versatility, and they possess indeed a rather effective esterase as well as other catalytic hydratase/hydrolase activitiesCitation9. Generally, other CA classes than the α-family do not possess esterase activity, although there are several erroneous reports of such an activity for β- and δ-CA enzymesCitation10, which have been shown by other groups to be artefactual dataCitation11. However, the η-CAs, present in protozoans belonging to the genus Plasmodium, PfaCACitation12, which have originally been annotated as being α-CAs, are known to possess esterase activity with 4-nitrophenyl acetate as substrateCitation13. They were subsequently shown to represent a new CA family, the η-class, and also proposed as a potential anti-malarial drug targetCitation12,Citation14. However, apart the initial reports from Krungkrai’s groupCitation13, which undoubtedly showed that PfaCA has esterase activity with 4-nitrophenyl acetate as substrate, and that this activity is potently inhibited by primary sulphonamidesCitation13, the main class of zinc-binding CAIsCitation15, no detailed such studies on this enzyme were performed. It should be stressed that after we showed that PfaCA is not an α- but an η-CACitation12, a multitude of sulphonamide and anion inhibitors of this enzyme (both for a truncated as well as for a longer form of it) have been detected, some with potency in the low nanomolar range (for the sulphonamides and their derivatives)Citation16.
Here we show that coumarins indeed act as PfaCA inhibitors, which is only possible due to the esterase activity of PfaCA, the prototypical η-class CA. In a small series of simple such derivatives, inhibition constants in the micromolar range against PfaCA were detected, and, more interestingly, many of the investigated coumarins were more effective protozoan enzyme inhibitors compared to their activity on the off-target human isoforms hCA I and II.
2. Materials and methods
2.1. Enzymology and CA activity and inhibition measurements
The CA-catalysed CO2 hydration activity has been measured with an Applied Photophysics stopped-flow instrumentCitation17. The used pH indicator was phenol red (at a concentration of 0.2 mM), working at the absorbance maximum of 557 nm. 10 mM HEPES (pH 7.4) was employed as a buffer, in the presence of 10 mM NaClO4 to maintain the ionic strength constant. The initial rates of the CA-catalysed CO2 hydration reaction were followed up for a period of 10–100 s. The substrate CO2 concentrations ranged from 1.7 to 17 mM for determining the inhibition constants. For each inhibitor, at least six traces of the initial 5–10% of the reaction were used to determine the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitors (10 mM) were prepared in distilled-deionized water with maximum 5% DMSO, and dilutions up to 10 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 1–6 h prior to the 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 previouslyCitation18–20, and represent the mean from at least three different determinations. The PfaCA concentration in the assay system was 12.38 nM. The human/protozoan enzymes were recombinant proteins obtained in-house, as described earlierCitation12,Citation16.
2.2. Chemistry
Coumarins 1–14, buffers, acetazolamide AAZ and other reagents were of >99% purity and were commercially available from Sigma-Aldrich (Milan, Italy).
3. Results and discussion
As mentioned in the introductory part, Krungkrai’s group first report that the Plasmodium falciparum genome encodes for CAs, which have been assigned to the α-classCitation13. In these initial studies, the esterase activity of such an enzyme, later denominated PfaCACitation12 has been observed, working with 4-nitrophenyl acetate as substrate, and indeed, the enzyme showed a significant such activity, which has been potently inhibited by primary sulphonamides and their isosteresCitation12,Citation14,Citation16, that are among the most investigated classes of CAIsCitation15.
A closer look at the amino acid sequence of PfaCA and orthologs from other Plasmodium species, allowed us to observe that these enzymes do not possess the three His ligands that coordinate the Zn(II) ion in all α-CAsCitation21, but instead the metal ion (which is crucial for catalysis) was proposed to be coordinated by two His and one Gln residuesCitation12. Indeed, a homology modelling study allowed us to propose the partial structure of the enzymeCitation12, which could not be modelled entirely as the enzyme used was a truncated form, but part of the active site and especially the metal ion and its ligands could be clearly modelled and are shown in .
Figure 1. Homology modelling and coordination of the zinc ion in the active site of PfaCA. The zinc ion (central grey sphere) is coordinated by the imidazole moieties of residues His299, His301 and the nitrogen from the CONH2 moiety of Gln320Citation12. The numbering of the amino acid residues is not shown for the sake of simplicity, but the 61 amino acid residues insertion which could not be modelled is highlighted in blue. The protein backbone is shown in green.
Although no X-ray crystallographic data were obtained so far for PfaCA, a previous study from Christianson’s group showed that mutating the His zinc ligands from the human isoform hCA II, such as for example the His119Gln substitution, leads to an enzyme that has the zinc coordination pattern presented in for PfaCA, and this enzyme also preserves its catalytic activity for the CO2 hydration reactionCitation22.
Such data prompted us to investigate the possible inhibitory activity of coumarins against PfaCA, which as mentioned above, must be hydrolysed by the esterase activity of the enzyme in order to generate the active inhibitorCitation1.
The simple mono- and di-substituted coumarins incorporating hydroxyl, amino, ketone or carboxylic acid ester moieties in various positions of the ring system of types 1–14 included in this study are shown in , together with their inhibitory data against PfaCA and two off-target human isoforms, hCA I and II. The following structure-activity relationship (SAR) can be observed from the above data:
Table 1. Inhibition data of hCA I and II and protozoan enzyme PfaCA with coumarins 1–14 and acetazolamide (AAZ) as standard drug by a stopped-flow CO2 hydrase assayCitation17.
the most effective PfaCA inhibitors in the investigated series were 2, 3, 6, 8 and 9, which showed inhibition constants ranging between 17.3 and 35.8 µM. The presence of OH moieties in positions 6- or 7- of the coumarin ring led to the most effective inhibitors (2 and 3, KIs of 17.3 – 20.4 µM), whereas amino, diethylamino, or methylketone groups (present in compounds 6, 8 and 9) led to slightly less effective PfaCA inhibitors. The presence of substituents in position 4 of the coumarin ring led to a decrease of potency for the methyl-containing such derivatives (6 and 8), which was even more accentuated for the when CF3 (derivative 7) or ethoxycarbonylmethyl (derivative 11) groups were present.
Medium potency PfaCA inhibition was observed with the following coumarins investigated here: 1, 4, 5, 7, 10 and 12, which had KIs of 46.9 – 90.5 µM (). The unsubstituted coumarin 1 is thus a medium potency-weak inhibitor (KI of 69.4 µM) but minor structural changes, such as the introduction of an OH group in positions 6 or 7, as shown above, drastically increase the inhibitory potency (derivatives 2 and 3 discussed above). However, the isomers with the OH group in positions 3 and 4 (compounds 4 and 5) showed a decrease of the inhibitory properties against PfaCA (KIs of 74.4 – 90.5 µM), demonstrating that these positions should be not substituted even with compact groups in order to obtain effective inhibitors. The same is true when a methyl is present in position 2, with compound 10 being 2.3 times a less effective PfaCA inhibitor compared to the de-methylated analog 3.
The least effective PfaCA inhibitors were 11, 13 and 14, which showed KIs of 311.0–455.0 µM (). These compounds incorporate bulkier moieties in positions 2 or 3 of the coumarin ring compared to the other derivatives included in the study, clearly demonstrating that the best activity is probably obtained when the lactone ring is unsubstituted. Modifications leading to effective inhibitors should thus consider substitution patterns in positions 6, 7 and 8 of the coumarin. This situation was in fact observed also for the inhibition of the human CA isoforms hCA I-XIV already in the first studies in which coumarins were reported as CAIsCitation1,Citation2.
The investigated coumarins were rather ineffective inhibitors of the human isoforms hCA I and II; with KIs in the range of 137.0–948.9 µM against hCA I and of KIs of 296.5–961.2 µM against hCA II. This is a relevant observation, as it demonstrates that the parasite enzyme is more inhibited than the human CAs included in the study.
4. Conclusions
This is the first study in which the inhibitory effects of coumarins against a non-α-CA are demonstrated. In a small series of mono- and di-substituted coumarins incorporating various substituents (OH, amino, Me, CF3, ketone, ethyl ester, etc.) and diverse substitution patterns, we demonstrate micromolar inhibition against PfaCA, a pathogen enzyme from the malaria provoking parasite P. falciparum. The SAR for obtaining effective PfaCA inhibitors is rather obvious, with the most effective compound having no substituents on the lactone ring and OH, amine or ketone groups in positions 6, 7 or 8 of the second ring. The study thus confirms that η-CAs possess esterase activity and that coumarins effectively inhibit this enzyme. Elaboration of the simple coumarin scaffolds investigated here may probably lead to more effective, presumably nanomolar PfaCA inhibitors, which might constitute interesting anti-malarial drug candidates.
Disclosure statement
CT Supuran is Editor-in-Chief of the Journal of Enzyme Inhibition and Medicinal Chemistry. He was not involved in the assessment, peer review, or decision-making process of this paper. The authors have no relevant affiliations of financial involvement with any organisation or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
Additional information
Funding
References
- (a) Maresca A, Temperini C, Vu H, et al. Non-zinc mediated inhibition of carbonic anhydrases: coumarins are a new class of suicide inhibitors. J Am Chem Soc 2009; 131:3057–62. (b) Maresca A, Temperini C, Pochet L, et al. Deciphering the mechanism of carbonic anhydrase inhibition with coumarins and thiocoumarins. J Med Chem 2010;53:335–44.
- (a) Maresca A, Supuran CT. Coumarins incorporating hydroxy- and chloro-moieties selectively inhibit the transmembrane, tumor-associated carbonic anhydrase isoforms IX and XII over the cytosolic ones I and II . Bioorg Med Chem Lett 2010; 20:4511–4. (b) Maresca A, Scozzafava A, Supuran CT. 7,8-Disubstituted- but not 6,7-disubstituted coumarins selectively inhibit the transmembrane, tumor-associated carbonic anhydrase isoforms IX and XII over the cytosolic ones I and II in the low nanomolar/subnanomolar range. Bioorg Med Chem Lett 2010; 20:7255–8. (c) Sharma A, Tiwari M, Supuran CT. Novel coumarins and benzocoumarins acting as isoform-selective inhibitors against the tumor-associated carbonic anhydrase IX. J Enzyme Inhib Med Chem 2014;29:292–6. (d) Touisni N, Maresca A, McDonald PC, et al. Glycosyl coumarin carbonic anhydrase IX and XII inhibitors strongly attenuate the growth of primary breast tumors. J Med Chem 2011;54:8271–7. (e) Fuentes-Aguilar A, Merino-Montiel P, Montiel-Smith S, et al. 2-Aminobenzoxazole-appended coumarins as potent and selective inhibitors of tumour-associated carbonic anhydrases. J Enzyme Inhib Med Chem 2022;37:168–77.
- (a) Petreni A, Osman SM, Alasmary FA, et al. Binding site comparison for coumarin inhibitors and amine/amino acid activators of human carbonic anhydrases. Eur J Med Chem 2021;226:113875. (b) Supuran CT. Coumarin carbonic anhydrase inhibitors from natural sources. J Enzyme Inhib Med Chem 2020;35:1462–70. (c) Supuran CT. Multitargeting approaches involving carbonic anhydrase inhibitors: hybrid drugs against a variety of disorders. J Enzyme Inhib Med Chem 2021;36:1702–14.
- (a) Fois B, Distinto S, Meleddu R, et al. Coumarins from Magydaris pastinacea as inhibitors of the tumour-associated carbonic anhydrases IX and XII: isolation, biological studies and in silico evaluation. J Enzyme Inhib Med Chem 2020;35:539–48. (b) Melis C, Distinto S, Bianco G, et al. Targeting tumor associated carbonic anhydrases IX and XII: highly isozyme selective coumarin and psoralen inhibitors. ACS Med Chem Lett 2018;9:725–9. (c) Meleddu R, Deplano S, Maccioni E, et al. Selective inhibition of carbonic anhydrase IX and XII by coumarin and psoralen derivatives. J Enzyme Inhib Med Chem 2021;36:685–92.
- (a) Mussi S, Rezzola S, Chiodelli P, et al. Antiproliferative effects of sulphonamide carbonic anhydrase inhibitors C18, SLC-0111 and acetazolamide on bladder, glioblastoma and pancreatic cancer cell lines. J Enzyme Inhib Med Chem 2022;37:280–6. (b) Supuran CT. Carbonic anhydrase inhibitors: an update on experimental agents for the treatment and imaging of hypoxic tumors. Expert Opin Investig Drugs 2021;30:1197–208. (c) Chen F, Licarete E, Wu X, et al. Pharmacological inhibition of carbonic anhydrase IX and XII to enhance targeting of acute myeloid leukaemia cells under hypoxic conditions. J Cell Mol Med 2021;25:11039–52. (d) McDonald PC, Chia S, Bedard PL, et al. A phase 1 study of SLC-0111, a novel inhibitor of carbonic anhydrase IX, in patients with advanced solid tumors. Am J Clin Oncol 2020;43:484–90.
- (a) Supuran CT. Novel carbonic anhydrase inhibitors. Future Med Chem 2021;13:1935–7. (b) Chafe SC, Vizeacoumar FS, Venkateswaran G, et al. Genome-wide synthetic lethal screen unveils novel CAIX-NFS1/xCT axis as a targetable vulnerability in hypoxic solid tumors. Sci Adv 2021;7:eabj0364. (c) Berrino E, Michelet B, Martin-Mingot A, et al. Modulating the efficacy of carbonic anhydrase inhibitors through fluorine substitution. Angew Chem Int Ed Engl 2021; 60:23068–82. (d) Supuran CT. Emerging role of carbonic anhydrase inhibitors. Clin Sci 2021;135:1233–49.
- (a) Alterio V, Langella E, Buonanno M, et al. Zeta-carbonic anhydrases show CS2 hydrolase activity: a new metabolic carbon acquisition pathway in diatoms? Comput Struct Biotechnol J 2021;19:3427–36. (b) Urbański LJ, Di Fiore A, Azizi L, et al. Biochemical and structural characterisation of a protozoan beta-carbonic anhydrase from Trichomonas vaginalis. J Enzyme Inhib Med Chem 2020;35:1292–9. (c) Amedei A, Capasso C, Nannini G, Supuran CT. Microbiota, bacterial carbonic anhydrases, and modulators of their activity: links to human diseases? Mediators Inflamm 2021; 2021:6926082.
- (a) Nocentini A, Supuran CT, Capasso C. An overview on the recently discovered iota-carbonic anhydrases. J Enzyme Inhib Med Chem 2021;36:1988–95. (b) Jensen EL, Maberly SC, Gontero B. Insights on the functions and ecophysiological relevance of the diverse carbonic anhydrases in microalgae. Int J Mol Sci 2020; 21:2922. (c) Jensen EL, Receveur-Brechot V, Hachemane M, et al. Structural contour map of the iota carbonic anhydrase from the diatom Thalassiosira pseudonana using a multiprong approach. Int J Mol Sci 2021;22:8723.
- (a) Pocker Y, Stone JT. The catalytic versatility of erythrocyte carbonic anhydrase. VI. Kinetic studies of noncompetitive inhibition of enzyme-catalyzed hydrolysis of p-nitrophenyl acetate. Biochemistry 1968;7:2936–45. (b) Pocker Y, Stone JT. The catalytic versatility of erythrocyte carbonic anhydrase. VII. Kinetic studies of esterase activity and competitive inhibition by substrate analogs. Biochemistry 1968;7:3021–31. (c) Pocker Y, Guilbert LJ. Catalytic versatility of erythrocyte carbonic anhydrase. Kinetic studies of the enzyme-catalyzed hydrolysis of methyl pyridyl carbonates. Biochemistry 1972; 11:180–90. (d) Pocker Y, Tanaka N. Inhibition of carbonic anhydrase by anions in the carbon dioxide-bicarbonate system. Science 1978;199:907–9. (e) Supuran CT, Conroy CW, Maren TH. Is cyanate a carbonic anhydrase substrate? Proteins 1997;27:272–8. (f) Nocentini A, Angeli A, Carta F, et al. Reconsidering anion inhibitors in the general context of drug design studies of modulators of activity of the classical enzyme carbonic anhydrase. J Enzyme Inhib Med Chem 2021;36:561–80.
- (a) Kaul T, Reddy PS, Mahanty S, et al. Biochemical and molecular characterization of stress-induced β-carbonic anhydrase from a C(4) plant, Pennisetum glaucum. J Plant Physiol 2011; 168:601–10. (b) Lee RB, Smith JA, Rickaby RE. Cloning, expression and characterization of the δ-carbonic anhydrase of Thalassiosira weissflogii (Bacillariophyceae). J Phycol 2013;49:170–7.
- (a) Del Prete S, Vullo D, De Luca V, et al. Biochemical characterization of the δ-carbonic anhydrase from the marine diatom Thalassiosira weissflogii, TweCA. J Enzyme Inhib Med Chem 2014; 29:906–11. (b) Innocenti A, Supuran CT. Paraoxon, 4-nitrophenyl phosphate and acetate are substrates of α- but not of β-, γ- and ζ-carbonic anhydrases. Bioorg Med Chem Lett 2010;20:6208–12. (c) Innocenti A, Scozzafava A, Parkkila S, et al. Investigations of the esterase, phosphatase, and sulfatase activities of the cytosolic mammalian carbonic anhydrase isoforms I, II, and XIII with 4-nitrophenyl esters as substrates. Bioorg Med Chem Lett 2008;18:2267–71. (d) Lopez M, Vu H, Wang CK, et al. Promiscuity of carbonic anhydrase II. Unexpected ester hydrolysis of carbohydrate-based sulfamate inhibitors. J Am Chem Soc 2011;133:18452–62.
- (a) Del Prete S, De Luca V, De Simone G, et al. Cloning, expression and purification of the complete domain of the η-carbonic anhydrase from Plasmodium falciparum. J Enzyme Inhib Med Chem 2016;31:54–9. (b) De Simone G, Di Fiore A, Capasso C, Supuran CT. The zinc coordination pattern in the η-carbonic anhydrase from Plasmodium falciparum is different from all other carbonic anhydrase genetic families. Bioorg Med Chem Lett 2015;25:1385–9. (c) Del Prete S, Vullo D, Fisher GM, et al. Discovery of a new family of carbonic anhydrases in the malaria pathogen Plasmodium falciparum-the η-carbonic anhydrases. Bioorg Med Chem Lett 2014;24:4389–96. (d) Del Prete S, De Luca V, Supuran CT, Capasso C. Protonography, a technique applicable for the analysis of η-carbonic anhydrase activity. J Enzyme Inhib Med Chem 2015;30:920–4.
- (a) Reungprapavut S, Krungkrai SR, Krungkrai J. Plasmodium falciparum carbonic anhydrase is a possible target for malaria chemotherapy. J Enzyme Inhib Med Chem 2004;19:249–56. (b) Krungkrai J, Scozzafava A, Reungprapavut S, et al. Carbonic anhydrase inhibitors. Inhibition of Plasmodium falciparum carbonic anhydrase with aromatic sulfonamides: towards antimalarials with a novel mechanism of action? Bioorg Med Chem 2005;13:483–9. (c) Krungkrai J, Supuran CT. The alpha-carbonic anhydrase from the malaria parasite and its inhibition. Curr Pharm Des 2008;14:631–40.
- (a) Supuran CT, Capasso C. The η-class carbonic anhydrases as drug targets for antimalarial agents. Expert Opin Ther Targets 2015;19:551–63. Apr (b) D'Ambrosio K, Supuran CT, De Simone G. Are carbonic anhydrases suitable targets to fight protozoan parasitic diseases? Curr Med Chem 2018;25:5266–78.
- (a) Briganti F, Pierattelli R, Scozzafava A, Supuran CT. Carbonic anhydrase inhibitors. Part 37. Novel classes of carbonic anhydrase inhibitors and their interaction with the native and cobalt-substituted enzyme: kinetic and spectroscopic investigations. Eur J Med Chem 1996;31:1001–10. (b) Pastorekova S, Casini A, Scozzafava A, et al. Carbonic anhydrase inhibitors: the first selective, membrane-impermeant inhibitors targeting the tumor-associated isozyme IX. Bioorg Med Chem Lett 2004;14:869–73. (c) Vullo D, Voipio J, Innocenti A, et al. Carbonic anhydrase inhibitors. Inhibition of the human cytosolic isozyme VII with aromatic and heterocyclic sulfonamides. Bioorg Med Chem Lett 2005;15:971–6. (d) Gieling RG, Babur M, Mamnani L, et al. Antimetastatic effect of sulfamate carbonic anhydrase IX inhibitors in breast carcinoma xenografts. J Med Chem 2012;55:5591–600. (e) Carta F, Supuran CT, Scozzafava A. Sulfonamides and their isosters as carbonic anhydrase inhibitors. Future Med Chem 2014;6:1149–65.
- (a) Angeli A, Pinteala M, Maier SS, et al. Inhibition of α-, β-, γ-, δ-, ζ- and η-class carbonic anhydrases from bacteria, fungi, algae, diatoms and protozoans with famotidine. J Enzyme Inhib Med Chem 2019; 34:644–50. (b) Del Prete S, Vullo D, De Luca V, et al. Cloning, expression, purification and sulfonamide inhibition profile of the complete domain of the η-carbonic anhydrase from Plasmodium falciparum. Bioorg Med Chem Lett 2016;26:4184–90. (c) Del Prete S, Vullo D, De Luca V, et al. Anion inhibition profiles of the complete domain of the η-carbonic anhydrase from Plasmodium falciparum. Bioorg Med Chem 2016;24:4410–4. (d) Vullo D, Del Prete S, Fisher GM, et al. Sulfonamide inhibition studies of the η-class carbonic anhydrase from the malaria pathogen Plasmodium falciparum. Bioorg Med Chem 2015;23:526–31. (e) Krungkrai J, Krungkrai SR, Supuran CT. Carbonic anhydrase inhibitors: inhibition of Plasmodium falciparum carbonic anhydrase with aromatic/heterocyclic sulfonamides-in vitro and in vivo studies. Bioorg Med Chem Lett 2008; 18:5466–71.
- Khalifah RG. The carbon dioxide hydration activity of carbonic anhydrase. I. Stop-flow kinetic studies on the native human isoenzymes B and C. J Biol Chem 1971; 246:2561–73.
- (a) Sarikaya SB, Gülçin I, Supuran CT. Carbonic anhydrase inhibitors: inhibition of human erythrocyte isozymes I and II with a series of phenolic acids. Chem Biol Drug Des 2010 May;75:515–20. (b) Yıldırım A, Atmaca U, Keskin A, et al. N-Acylsulfonamides strongly inhibit human carbonic anhydrase isoenzymes I and II. Bioorg Med Chem 2015;23:2598–605. (c) Innocenti A, Gülçin I, Scozzafava A, Supuran CT. Carbonic anhydrase inhibitors. Antioxidant polyphenols effectively inhibit mammalian isoforms I-XV. Bioorg Med Chem Lett 2010;20:5050–3.
- (a) Zimmerman SA, Ferry JG, Supuran CT. Inhibition of the archaeal beta-class (Cab) and gamma-class (Cam) carbonic anhydrases. Curr Top Med Chem 2007;7:901–8. (b) Maresca A, Carta F, Vullo D, Supuran CT. Dithiocarbamates strongly inhibit the β-class carbonic anhydrases from Mycobacterium tuberculosis. J Enzyme Inhib Med Chem 2013;28:407–11.
- (a) Winum JY, Temperini C, El Cheikh K, et al. Carbonic anhydrase inhibitors: clash with Ala65 as a means for designing inhibitors with low affinity for the ubiquitous isozyme II, exemplified by the crystal structure of the topiramate sulfamide analogue. J Med Chem 2006;49:7024–31. (b) Oztürk Sarikaya SB, Topal F, Sentürk M, et al. In vitro inhibition of α-carbonic anhydrase isozymes by some phenolic compounds. Bioorg Med Chem Lett 2011;21:4259–62.
- (a) Supuran CT. Structure and function of carbonic anhydrases. Biochem J 2016;473:2023–32. (b) Mishra CB, Tiwari M, Supuran CT. Progress in the development of human carbonic anhydrase inhibitors and their pharmacological applications: where are we today? Med Res Rev 2020; 40:2485–565. (c) Supuran CT. Carbonic anhydrases: novel therapeutic applications for inhibitors and activators. Nat Rev Drug Discov 2008;7:168–81.
- Lesburg CA, Huang C, Christianson DW, Fierke CA. Histidine -> carboxamide ligand substitutions in the zinc binding site of carbonic anhydrase II alter metal coordination geometry but retain catalytic activity. Biochemistry 1997;36:15780–91.