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Acyl selenoureido benzensulfonamides show potent inhibitory activity against carbonic anhydrases from the pathogenic bacterium Vibrio cholerae
Bioorganic Chemistry 75 (2017) 170–172
Contents lists available at ScienceDirect
Bioorganic Chemistry journal homepage: www.elsevier.com/locate/bioorg
Short communication
Acyl selenoureido benzensulfonamides show potent inhibitory activity against carbonic anhydrases from the pathogenic bacterium Vibrio cholerae Andrea Angeli a, Ghulam Abbas a,b, Sonia Del Prete c, Fabrizio Carta a, Clemente Capasso c, Claudiu T. Supuran a,⇑ a
Università degli Studi di Firenze, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy Department of Biological Sciences and Chemistry, University of Nizwa, Birkat Al-Mauz, P.O.Box 33, Nizwa 616, Oman c Istituto di Bioscienze e Biorisorse, CNR, Via Pietro Castellino 111, 80131 Napoli, Italy b
a r t i c l e
i n f o
Article history: Received 23 August 2017 Revised 19 September 2017 Accepted 20 September 2017 Available online 21 September 2017 Keywords: Carbonic anhydrase Inhibitors Metalloenzymes Selenium Acylseleno-ureido Vibrio cholerae
a b s t r a c t A series of acyl selenoureido benzensulfonamides was evaluated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors against two Vibrio cholerae such enzymes (VchCAa over VchCAb) belonging to the a- and bclasses, potential novel targets for anti-infective drugs development. These compounds showed strong inhibitory action against VchCAa over VchCAb and excellent selectivity over the human (h) off-target isoforms hCA I and II. Identification of potent and possibly selective inhibitors of VchCAa and/or VchCAb over the human counterparts may lead to pharmacological tools useful for understanding the physiological role(s) of these under-investigated proteins, possibly involved in the virulence of the bacterium and colonization of the host in bicarbonate rich regions of the gastro-intestinal tract. Ó 2017 Elsevier Inc. All rights reserved.
1. Introduction Vibrio cholerae, the etiological agent of cholera, is a Gramnegative bacterium provoking serious or even fatal disease if untreated, due to the massive loss of fluids after infection [1]. The number of reported cholera cases remains high over the past decades. WHO reported 172,454 cases in 42 countries during 2015, which also included 1304 deaths [2]. In addition, the infectious diseases are the second-leading cause of death in the world and the emergence of antibiotic-resistant microorganisms is an inevitable and widespread phenomenon, inherent to most clinically used drugs [3–8] Recently, carbonic anhydrases (CAs, EC 4.2.1.1) have started to be investigated in detail in microorganisms, in the search for anti-infectives with a novel mechanism of action [8,9]. It has been indeed demonstrated that in many pathogenic microorganisms the CAs are essential for their life cycle and that inhibiting this enzyme leads to growth impairment of the pathogen [5–10]. The CAs from the bacterial pathogen V. cholerae were described by our group: an a-CA, denominated VchCAa was first characterized [9a], which similar to the other a-CA investigated so far, has ⇑ Corresponding author. E-mail address: [email protected] (C.T. Supuran). https://doi.org/10.1016/j.bioorg.2017.09.016 0045-2068/Ó 2017 Elsevier Inc. All rights reserved.
three His ligands which coordinate the Zn(II) ion which is crucial for catalysis. A b-class CA was subsequently cloned, purified and characterized from this pathogen, VchCAb [9b], which together with the a-CA produce bicarbonate by catalyzing the hydration of CO2 [9,10]. It has been demonstrated that bicarbonate plays a crucial role as a virulence factor for V. cholerae [11]. Thus, VchCAa/b have been suggested as potential targets for antibiotic development [9,10]. During the last decades, new selenium containing scaffolds have successfully been designed and screened as antimicrobial agents [12–14]. However, until now few efforts have been made for the applications of organoselenium derivatives in enzyme inhibition studies [15,16], and continuing our efforts in the development of organoselenium sulfonamides CA inhibitors (CAIs), we investigate here acyl selenoureido benzenesulfonamides as inhibitors of VchCAa and VchCAb.
2. Results and discussion 2.1. Chemistry Commercially available acyl chloride 1a–h, have been used for preparing the corresponding acyl seleno ureido derivatives 6–18,
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as reported earlier by our group [16] (Scheme 1). Our main interest was to investigate structure–activity relationship related to the inhibition of the two pathogenic enzymes from Vibrio cholerae, VchCAa and VchCAb. The rationale of introducing the CO-NH-CSe-NH moiety in sulfonamide CAIs is that the many heteroatoms present in this functionality may interact in a favourable manner with amino acid residues from the enzymes active sites, leading thus to more effective inhibitors, as shown earlier for other CAs [16]. 2.2. Carbonic anhydrase inhibition The inhibition studies of VchCAa and VchCAb with compounds 6–18 were performed in order to detect possible candidates for antiinfective studies. We tested these compounds for their inhibitory activity against the two physiologically relevant hCA isoforms I, II (off-targets), and the bacterial enzyme VchCAa, VchCAb by means of the stopped-flow carbon dioxide hydration assay [17]. These activities were compared to those of the standard, clinically used CAI acetazolamide (AAZ) (Table 1). The selectivity ratios for the inhibition of the bacterial versus the human isoform hCA II (the physiologically dominant one) with these compounds are also included in Table 1. The following structure-activity-relationship (SAR) may be noted regarding the inhibition data of Table 1: Acyl selenoureido compounds 6–18 exhibited a highly potent inhibitory activity towards VchCAa, with inhibition constants ranging in the sub-nanomolar range, except for derivative 18, which was active in the low nanomolar range (overall, the KIs ranged between 0.3 and 8.4 nM). This suggests that the placement of the sulfonamide moiety in these selenium-containing analogs, leads to an excellent inhibitory potency against VchCAa, rarely observed with other CAIs of the sulfonamide type [9,10]. In fact, all these derivatives (except 18) were so potent CAIs that a proper SAR is impossible to draw. As mentionated above, only the orthosubstituted derivative 18 showed a somehow decreased activity compared to the other sulfonamides, but this compound is as active as AAZ in our assays (Table 1). The introduction of small substituents on the acyl scaffold incorporating 4-ethylamino benzensulfonamide (6 9) did not influence significantly the potency
Table 1 Inhibition data against human (h) isoforms hCA I, II (cytosolic enzymes), and bacterial enzyme VchCAa, VchCAb of derivates 6–18 and acetazolamide AAZ by a stopped flow CO2 hydrase assay [17]. KI (nM)*
Selectivity ratioa
Cmp
hCA I
hCA II
VchCAa
VchCAb
hCA II/VchCAa
6 7 8 9 10 11 12 13 14 15 16 17 18 AAZ
65.6 514.9 3745.9 523.7 73.8 8702.0 85.7 85.7 734.2 521.2 81.6 9381.1 61.5 250
18.7 21.8 9533.7 26.9 9.5 402.4 0.7 9.1 48.9 247.4 6.6 6072.3 66.9 12.1
0.67 0.77 0.95 0.30 0.68 0.58 0.88 0.66 0.30 0.67 0.36 0.74 8.4 6.8
5574 5335 5989 6777 6620 5487 6821 5242 8057 8771 7127 6378 4530 451
27.9 28.3 10035.4 89.7 14 693.8 0.79 13.8 16.3 369.2 18.3 8205.8 8 1.8
* Mean from 3 different assays, by a stopped flow technique (errors were in the range of ±5–10% of the reported values). a Selectivity as determined by the ratio of Ki for hCA II isoform relative to VchCAa.
for inhibition for VchCAa, although, the benzodioxole scaffold (9) showed the best activity with a KI of 0.3 nM. An interesting inhibition profile was observed for compounds 8 and 17. The presence of another phenyl moiety as in 8, or two CF3 groups in the acyl scaffold, as in 17, decreased the inhibition potency for all humans and b-CA isoforms but, did not influence VchCAa inhibition. Thus, these compounds showed excellent selectivity, by over a thousand folds, for this isoform. Indeed, unlike the standard drug acetazolamide (AAZ), acyl selenoureido sulfonamides reported here were generally poor inhibitors against VchCAb, showing inhibition constants in high micromolar range (KIs of 4.53–8.77 mM)ab. On the other hand, all the synthesised compounds (except for derivative 12) were selective inhibitors for the bacterial VchCAa isoform over the human enzymes hCA I and II, as well as VchCAb. It is well known [18] that sulfonamide CAIs generally show a higher affinity for a-class than for b-class enzymes, for reasons connected to the active site architecture of these enzymes. Indeed, a-CAs possess a
Scheme 1. General procedure for the synthesis of acyl selenoureido compounds 6–18 [16].
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wider and deeper active site [18a–c] compared to the active site of the b-CAs, which is a rather long but less widely opened channel [18d]. This is well reflected for VchCA b, an enzyme for which the X-ray crystal structure was recently reported. It is a tetramer with four identical active sites having the shape of an elongated channel [18d]. Probably the oligomeric organization of this tetrameric enzyme also interferes with the binding of sulfonamides, which may explain the differences of activity observed between VchCAa and VchCAb inhibition with the compounds investigated here. 3. Conclusions We have investigated a series of acyl selenoureido benzenesulfonamides for the inhibition of two bacterial CA isoforms from the human pathogen Vibrio cholerae. Excellent inhibitory activity, in the subnanomolar range, was observed against VchCAa whereas the b class enzyme was much less inhibited (in the micromolar range). These compounds were also VchCAa-selective inhibitors over the human offtarget enzymes hCA I, II. Identification of potent and possibly isoform-selective inhibitors of VchCAa may lead to pharmacological tools useful for understanding the physiological role(s) of these under-investigated enzymes. 4. Experimental part 4.1. Chemistry Compounds 6–18 were reported earlier by our group [16b]. 4.2. Carbonic anhydrase inhibition An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalyzed CO2 hydration activity [17]. 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 for the a-CAs) or TRIS (pH 8.3 for the b-CAs) as buffers, and 20 mM Na2SO4 (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 (0.1 mM) were prepared in distilled-deionized 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 non-linear least-squares methods using PRISM 3 and the Cheng–Prusoff equation, as reported earlier [19–21], and represent the mean from at least three different determinations. All CA isofoms were recombinant ones obtained in-house as reported earlier [19–21]. References [1] M. Kitaoka, S.T. Miyata, D. Unterweger, S. Pukatzki, J. Microbiol. 60 (2011) 397– 407. [2] Cholera Annual Report 2015, Weekly Epidemiological Record 23 September 2016; 91, 38: 433–440. available on line at http://www.who.int/wer/2016/ wer9138/en/.
[3] J. Sacarlal, A.Q. Nhacolo, B. Sigauque, et al., BMC Publ. Health 9 (2009) 67. [4] (a) C. Capasso, C.T. Supuran, Expert Opin. Ther. Pat. 23 (2013) 693–704; (b) C.T. Supuran, Biochem. J. 473 (2016) 2023–2032; (c) S. Del Prete, D. Vullo, S.M. Osman, Z. AlOthman, W.A. Donald, J.Y. Winum, C. T. Supuran, C. Capasso, Bioorg. Med. Chem. 25 (2017) 4800–4804; (d) D. Vullo, S. Del Prete, P. Di Fonzo, V. Carginale, W.A. Donald, C.T. Supuran, C. Capasso, Molecules 22 (2017) E421; (e) S. Del Prete, D. Vullo, P. Di Fonzo, S.M. Osman, Z. AlOthman, W.A. Donald, C. T. Supuran, C. Capasso, Bioorg. Med. Chem. Lett. 27 (2017) 490–495. [5] (a) S. Rusconi, A. Scozzafava, A. Mastrolorenzo, et al., Curr. Drug Targets Infect. Disord. 4 (2004) 339–355; (b) J.K. Modak, Y.C. Liu, M.A. Machuca, C.T. Supuran, A. Roujeinikova, PLoS One 10 (2015) e0127149; (c) C.T. Supuran, J. Enzyme Inhib. Med. Chem. 31 (2016) 345–360. [6] (a) C.T. Supuran, Nat. Rev. Drug Discov. 7 (2008) 168; (b) A. Innocenti, R.A. Hall, C. Schlicker, et al., Bioorg. Med. Chem. 17 (2009) 4503–4509; (c) C.T. Supuran, Expert Opin. Drug Discov. 12 (2017) 6–88. [7] (a) A. Maresca, A. Scozzafava, S. Kohler, et al., J. Inorg. Biochem. 110 (2012) 36– 39; (b) C.T. Supuran, Curr. Med. Chem. 19 (2012) 831–844; (c) C.T. Supuran, J. Enzyme Inhib. Med. Chem. 27 (2012) 759–772. [8] (a) C.T. Supuran, C. Capasso, Expert Opin. Ther. Targets 19 (2015) 551–563; (b) S. Del Prete, V. De Luca, C.T. Supuran, C. Capasso, J. Enzyme Inhib. Med. Chem. 30 (2015) 920; (c) C. Capasso, C.T. Supuran, J. Enzyme Inhib. Med. Chem. 30 (2015) 325–332; (d) C.T. Supuran, C. Capasso, J. Enzyme Inhib. Med. Chem. 31 (2016) 1254; (e) G.M. Buzas, C.T. Supuran, J. Enzyme Inhib. Med. Chem. 31 (2016) 527–533. [9] (a) S. Del Prete, V. De Luca, A. Scozzafava, V. Carginale, C.T. Supuran, C. Capasso, J. Enzyme Inhib. Med. Chem. 29 (2014) 23–27; (b) S. Del Prete, D. Vullo, V. De Luca, V. Carginale, M. Ferraroni, S.M. Osman, Z. AlOthman, C.T. Supuran, C. Capasso, Bioorg. Med. Chem. 24 (2016) 1115. [10] A.M. Alafeefy, M. Ceruso, A.-M.S. Al-Tamimi, S. Del Prete, C. Capasso, C.T. Supuran, Bioorg. Med. Chem. 22 (2014) 5133–5140. [11] (a) J.J. Thomson, J.H. Withey, J. Bacteriol. 196 (2014) 3872–3880; (b) B.H. Abuaita, J.H. Withey, Infect. Immun. 77 (2009) 4111–4120. [12] (a) R. Nozawa, T. Yokota, R. Fujimoto, Antimicrob. Agents Chemother. 33 (1989) 1388; (b) M. Alpegiani, A. Bedeschi, E. Perrone, G. Franceschi, Tetrahedron Lett. 27 (1986) 3041. [13] G.A. Brown, K.M. Anderson, M. Murray, T. Gallagher, N.J. Hales, Tetrahedron 56 (2000) 5579. [14] M. Koketsu, H. Ishihara, M. Hatsu, Res. Commun. Mol. Path. Pharmacol. 101 (1998) 179. [15] A. Angeli, D. Tanini, C. Viglianisi, L. Panzella, A. Capperucci, S. Menichetti, C.T. Supuran, Bioorg. Med. Chem. 17 (2017) 30326–30327. [16] (a) A. Angeli, T.S. Peat, G. Bartolucci, A. Nocentini, C.T. Supuran, F. Carta, Org. Biomol. Chem. 14 (2016) 11353; (b) A. Angeli, F. Carta, G. Bartolucci, C.T. Supuran, Bioorg. Med. Chem. 25 (2017) 3567–3573. [17] R.G. Khalifah, J. Biol. Chem. 246 (1971) 2561. [18] (a) V. Alterio, A. Di Fiore, K. D’Ambrosio, C.T. Supuran, G. De Simone, Chem. Rev. 112 (2012) 4421–4468; (b) F. Briganti, R. Pierattelli, A. Scozzafava, C.T. Supuran, Eur. J. Med. Chem. 31 (1996) 1001–1010; (c) C.T. Supuran, B.W. Clare, Eur. J. Med. Chem. 34 (1999) 41–50; (d) M. Ferraroni, S. Del Prete, D. Vullo, C. Capasso, C.T. Supuran, Acta Crystallogr. D Biol. Crystallogr. 71 (2015) 2449–2456. [19] (a) D. De Vita, A. Angeli, F. Pandolfi, M. Bortolami, R. Costi, R. Di Santo, E. Suffredini, M. Ceruso, S. Del Prete, C. Capasso, L. Scipione, C.T. Supuran, J. Enzyme Inhib. Med. Chem. 32 (2017) 798–804; (b) C. Melis, R. Meleddu, A. Angeli, S. Distinto, G. Bianco, C. Capasso, F. Cottiglia, R. Angius, C.T. Supuran, E. Maccioni, J. Enzyme Inhib. Med. Chem. 32 (2017) 68–73; (c) S. Angapelly, P.V. Ramya, A. Angeli, S.M. Monti, M. Buonanno, M. Alvala, C.T. Supuran, M. Arifuddin, Bioorg. Med. Chem. 25 (2017) 539–544. [20] (a) M. Abdoli, A. Angeli, M. Bozdag, F. Carta, A. Kakanejadifard, H. Saeidian, C.T. Supuran, J. Enzyme Inhib. Med. Chem. 32 (2017) 1071–1078; (b) C.B. Mishra, S. Kumari, A. Angeli, S.M. Monti, M. Buonanno, M. Tiwari, C.T. Supuran, J. Med. Chem. 60 (2017) 2456–2469; (c) A.A. Abdel-Aziz, A. Angeli, A.S. El-Azab, M.A. Abu El-Enin, C.T. Supuran, Bioorg. Med. Chem. 25 (2017) 1666–1671; (d) L. Puccetti, G. Fasolis, D. Vullo, Z.H. Chohan, A. Scozzafava, C.T. Supuran, Bioorg. Med. Chem. Lett. 15 (2005) 3096–3101. [21] (a) A. Mollica, M. Locatelli, G. Macedonio, S. Carradori, A.P. Sobolev, R.F. De Salvador, S.M. Monti, M. Buonanno, G. Zengin, A. Angeli, C.T. Supuran, J. Enzyme Inhib. Med. Chem. 31 (sup4) (2016) 1–6; (b) A. Scozzafava, L. Menabuoni, F. Mincione, C.T. Supuran, J. Med. Chem. 45 (2002) 1466–1476.
Contents lists available at ScienceDirect
Bioorganic Chemistry journal homepage: www.elsevier.com/locate/bioorg
Short communication
Acyl selenoureido benzensulfonamides show potent inhibitory activity against carbonic anhydrases from the pathogenic bacterium Vibrio cholerae Andrea Angeli a, Ghulam Abbas a,b, Sonia Del Prete c, Fabrizio Carta a, Clemente Capasso c, Claudiu T. Supuran a,⇑ a
Università degli Studi di Firenze, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy Department of Biological Sciences and Chemistry, University of Nizwa, Birkat Al-Mauz, P.O.Box 33, Nizwa 616, Oman c Istituto di Bioscienze e Biorisorse, CNR, Via Pietro Castellino 111, 80131 Napoli, Italy b
a r t i c l e
i n f o
Article history: Received 23 August 2017 Revised 19 September 2017 Accepted 20 September 2017 Available online 21 September 2017 Keywords: Carbonic anhydrase Inhibitors Metalloenzymes Selenium Acylseleno-ureido Vibrio cholerae
a b s t r a c t A series of acyl selenoureido benzensulfonamides was evaluated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors against two Vibrio cholerae such enzymes (VchCAa over VchCAb) belonging to the a- and bclasses, potential novel targets for anti-infective drugs development. These compounds showed strong inhibitory action against VchCAa over VchCAb and excellent selectivity over the human (h) off-target isoforms hCA I and II. Identification of potent and possibly selective inhibitors of VchCAa and/or VchCAb over the human counterparts may lead to pharmacological tools useful for understanding the physiological role(s) of these under-investigated proteins, possibly involved in the virulence of the bacterium and colonization of the host in bicarbonate rich regions of the gastro-intestinal tract. Ó 2017 Elsevier Inc. All rights reserved.
1. Introduction Vibrio cholerae, the etiological agent of cholera, is a Gramnegative bacterium provoking serious or even fatal disease if untreated, due to the massive loss of fluids after infection [1]. The number of reported cholera cases remains high over the past decades. WHO reported 172,454 cases in 42 countries during 2015, which also included 1304 deaths [2]. In addition, the infectious diseases are the second-leading cause of death in the world and the emergence of antibiotic-resistant microorganisms is an inevitable and widespread phenomenon, inherent to most clinically used drugs [3–8] Recently, carbonic anhydrases (CAs, EC 4.2.1.1) have started to be investigated in detail in microorganisms, in the search for anti-infectives with a novel mechanism of action [8,9]. It has been indeed demonstrated that in many pathogenic microorganisms the CAs are essential for their life cycle and that inhibiting this enzyme leads to growth impairment of the pathogen [5–10]. The CAs from the bacterial pathogen V. cholerae were described by our group: an a-CA, denominated VchCAa was first characterized [9a], which similar to the other a-CA investigated so far, has ⇑ Corresponding author. E-mail address: [email protected] (C.T. Supuran). https://doi.org/10.1016/j.bioorg.2017.09.016 0045-2068/Ó 2017 Elsevier Inc. All rights reserved.
three His ligands which coordinate the Zn(II) ion which is crucial for catalysis. A b-class CA was subsequently cloned, purified and characterized from this pathogen, VchCAb [9b], which together with the a-CA produce bicarbonate by catalyzing the hydration of CO2 [9,10]. It has been demonstrated that bicarbonate plays a crucial role as a virulence factor for V. cholerae [11]. Thus, VchCAa/b have been suggested as potential targets for antibiotic development [9,10]. During the last decades, new selenium containing scaffolds have successfully been designed and screened as antimicrobial agents [12–14]. However, until now few efforts have been made for the applications of organoselenium derivatives in enzyme inhibition studies [15,16], and continuing our efforts in the development of organoselenium sulfonamides CA inhibitors (CAIs), we investigate here acyl selenoureido benzenesulfonamides as inhibitors of VchCAa and VchCAb.
2. Results and discussion 2.1. Chemistry Commercially available acyl chloride 1a–h, have been used for preparing the corresponding acyl seleno ureido derivatives 6–18,
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as reported earlier by our group [16] (Scheme 1). Our main interest was to investigate structure–activity relationship related to the inhibition of the two pathogenic enzymes from Vibrio cholerae, VchCAa and VchCAb. The rationale of introducing the CO-NH-CSe-NH moiety in sulfonamide CAIs is that the many heteroatoms present in this functionality may interact in a favourable manner with amino acid residues from the enzymes active sites, leading thus to more effective inhibitors, as shown earlier for other CAs [16]. 2.2. Carbonic anhydrase inhibition The inhibition studies of VchCAa and VchCAb with compounds 6–18 were performed in order to detect possible candidates for antiinfective studies. We tested these compounds for their inhibitory activity against the two physiologically relevant hCA isoforms I, II (off-targets), and the bacterial enzyme VchCAa, VchCAb by means of the stopped-flow carbon dioxide hydration assay [17]. These activities were compared to those of the standard, clinically used CAI acetazolamide (AAZ) (Table 1). The selectivity ratios for the inhibition of the bacterial versus the human isoform hCA II (the physiologically dominant one) with these compounds are also included in Table 1. The following structure-activity-relationship (SAR) may be noted regarding the inhibition data of Table 1: Acyl selenoureido compounds 6–18 exhibited a highly potent inhibitory activity towards VchCAa, with inhibition constants ranging in the sub-nanomolar range, except for derivative 18, which was active in the low nanomolar range (overall, the KIs ranged between 0.3 and 8.4 nM). This suggests that the placement of the sulfonamide moiety in these selenium-containing analogs, leads to an excellent inhibitory potency against VchCAa, rarely observed with other CAIs of the sulfonamide type [9,10]. In fact, all these derivatives (except 18) were so potent CAIs that a proper SAR is impossible to draw. As mentionated above, only the orthosubstituted derivative 18 showed a somehow decreased activity compared to the other sulfonamides, but this compound is as active as AAZ in our assays (Table 1). The introduction of small substituents on the acyl scaffold incorporating 4-ethylamino benzensulfonamide (6 9) did not influence significantly the potency
Table 1 Inhibition data against human (h) isoforms hCA I, II (cytosolic enzymes), and bacterial enzyme VchCAa, VchCAb of derivates 6–18 and acetazolamide AAZ by a stopped flow CO2 hydrase assay [17]. KI (nM)*
Selectivity ratioa
Cmp
hCA I
hCA II
VchCAa
VchCAb
hCA II/VchCAa
6 7 8 9 10 11 12 13 14 15 16 17 18 AAZ
65.6 514.9 3745.9 523.7 73.8 8702.0 85.7 85.7 734.2 521.2 81.6 9381.1 61.5 250
18.7 21.8 9533.7 26.9 9.5 402.4 0.7 9.1 48.9 247.4 6.6 6072.3 66.9 12.1
0.67 0.77 0.95 0.30 0.68 0.58 0.88 0.66 0.30 0.67 0.36 0.74 8.4 6.8
5574 5335 5989 6777 6620 5487 6821 5242 8057 8771 7127 6378 4530 451
27.9 28.3 10035.4 89.7 14 693.8 0.79 13.8 16.3 369.2 18.3 8205.8 8 1.8
* Mean from 3 different assays, by a stopped flow technique (errors were in the range of ±5–10% of the reported values). a Selectivity as determined by the ratio of Ki for hCA II isoform relative to VchCAa.
for inhibition for VchCAa, although, the benzodioxole scaffold (9) showed the best activity with a KI of 0.3 nM. An interesting inhibition profile was observed for compounds 8 and 17. The presence of another phenyl moiety as in 8, or two CF3 groups in the acyl scaffold, as in 17, decreased the inhibition potency for all humans and b-CA isoforms but, did not influence VchCAa inhibition. Thus, these compounds showed excellent selectivity, by over a thousand folds, for this isoform. Indeed, unlike the standard drug acetazolamide (AAZ), acyl selenoureido sulfonamides reported here were generally poor inhibitors against VchCAb, showing inhibition constants in high micromolar range (KIs of 4.53–8.77 mM)ab. On the other hand, all the synthesised compounds (except for derivative 12) were selective inhibitors for the bacterial VchCAa isoform over the human enzymes hCA I and II, as well as VchCAb. It is well known [18] that sulfonamide CAIs generally show a higher affinity for a-class than for b-class enzymes, for reasons connected to the active site architecture of these enzymes. Indeed, a-CAs possess a
Scheme 1. General procedure for the synthesis of acyl selenoureido compounds 6–18 [16].
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wider and deeper active site [18a–c] compared to the active site of the b-CAs, which is a rather long but less widely opened channel [18d]. This is well reflected for VchCA b, an enzyme for which the X-ray crystal structure was recently reported. It is a tetramer with four identical active sites having the shape of an elongated channel [18d]. Probably the oligomeric organization of this tetrameric enzyme also interferes with the binding of sulfonamides, which may explain the differences of activity observed between VchCAa and VchCAb inhibition with the compounds investigated here. 3. Conclusions We have investigated a series of acyl selenoureido benzenesulfonamides for the inhibition of two bacterial CA isoforms from the human pathogen Vibrio cholerae. Excellent inhibitory activity, in the subnanomolar range, was observed against VchCAa whereas the b class enzyme was much less inhibited (in the micromolar range). These compounds were also VchCAa-selective inhibitors over the human offtarget enzymes hCA I, II. Identification of potent and possibly isoform-selective inhibitors of VchCAa may lead to pharmacological tools useful for understanding the physiological role(s) of these under-investigated enzymes. 4. Experimental part 4.1. Chemistry Compounds 6–18 were reported earlier by our group [16b]. 4.2. Carbonic anhydrase inhibition An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalyzed CO2 hydration activity [17]. 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 for the a-CAs) or TRIS (pH 8.3 for the b-CAs) as buffers, and 20 mM Na2SO4 (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 (0.1 mM) were prepared in distilled-deionized 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 non-linear least-squares methods using PRISM 3 and the Cheng–Prusoff equation, as reported earlier [19–21], and represent the mean from at least three different determinations. All CA isofoms were recombinant ones obtained in-house as reported earlier [19–21]. References [1] M. Kitaoka, S.T. Miyata, D. Unterweger, S. Pukatzki, J. Microbiol. 60 (2011) 397– 407. [2] Cholera Annual Report 2015, Weekly Epidemiological Record 23 September 2016; 91, 38: 433–440. available on line at http://www.who.int/wer/2016/ wer9138/en/.
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