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Received 00th January 20xx, Accepted 00th January 20xx DOI: 10.1039/x0xx00000x www.rsc.org/
HYDROXYLAMINE-O-SULFONAMIDE IS A VERSATILE LEAD COMPOUND FOR THE DEVELOPMENT OF CARBONIC ANHYDRASE INHIBITORS Anna Di Fiore,a† Alessandro Vergara,a,b Marco Caterino,b Vincenzo Alterio,a Simona M. Monti,a Joanna Ombouma,c Pascal Dumy,c Daniela Vullo,d Claudiu T. Supuran,d Jean-Yves Winum,c and Giuseppina De Simonea†
Hydroxylamine-O-sulfonamide, a molecule incorporating two zincbinding groups (ZBGs), has been investigated as carbonic anhydrase inhibitor (CAI) by means of kinetic, crystallographic and Raman spectroscopy studies, highlighting interesting results on its mechanism of action. These data can be exploited to design new effective and selective CAIs. Carbonic anhydrases (CAs) are ubiquitous metallo-enzymes which 1, 2 catalyze the reversible CO2 hydration reaction. These enzymes are widespread in all the kingdoms of life and are encoded by six evolutionarily unrelated gene families: α-, β-, γ-, δ-, ζ- and η-CAs.1, 3 Human (h) CAs belong to the α-class and are present in 15 isoforms, which differ by molecular features, oligomeric arrangement, cellular 1 localization, tissue distribution, and kinetic properties. Considering the crucial role of the CA catalyzed reaction, these enzymes are 4-8 involved in a variety of physiological/pathological processes, and consequently have lately become important targets for 1 pharmaceutical research. In recent years, numerous studies have been reported on CA inhibitors (CAIs). In particular, the elucidation of their mechanism of action, through the crystallographic analysis of several enzymeinhibitor adducts, allowed to determine the molecular basis of enzyme-inhibitor interactions.1 The most effective CAIs are sulfonamide/sulfamate-based molecules that interact with the enzyme active site coordinating the zinc ion and displacing the 1 water molecule/hydroxide ion present in the native enzyme; however, these molecules are generally poorly selective towards the different CA isoforms. For this reason, despite many encouraging results, new zinc binding groups (ZBGs) are continuously tested in order to advance upon the identification of
9
isoform selective CAIs.
In this paper, we report the synthesis, inhibition and structural studies of hydroxylamine-O-sulfonamide 1 (Figure 1), a molecule containing two ZBGs, namely the sulfonamide and hydroxylamine moieties. Our initial working hypothesis was that the concurrent presence of two functional groups could give an ambidentate character to this derivative. Our data show that compound 1 possesses an interesting inhibition profile against the different hCA isoforms as well as an intriguing binding mode. Indeed, it can bind the zinc ion of the CA active site with both ZBGs, thus suggesting that the derivatization both of the sulfonamide nitrogen and of the hydroxylamine moiety can be used to obtain new classes of 10 inhibitors.
Figure 1 Chemical structures of compounds 1–5. Compound 1 was synthesized in a two step synthesis starting from the commercially available N-Boc-hydroxylamine A as depicted in Scheme 1. Reaction of sulfamoylation of A with sulfamoyl chloride led quantitatively to the N-Boc-O-sulfamoyl hydroxylamine B. The latter was then reacted with a solution of TFA in dichloromethane to afford the hydroxylamine-O-sulfonamide 1 as trifluoroacetate salt (see Scheme 1 and Supplementary information for details).
a.
Istituto di Biostrutture e Bioimmagini-CNR, via Mezzocannone 16, 80134 Napoli, Italy. Department of Chemical Sciences, University of Naples Federico II, Napoli, Via Cinthia 80126, Italy. c. Institut des Biomolécules Max Mousseron (IBMM) UMR 5247 CNRS, ENSCM, Université de Montpellier, Bâtiment de Recherche Max Mousseron, Ecole Nationale Supérieure de Chimie de Montpellier, 8 rue de l’Ecole Normale, 34296 Montpellier Cedex, France. d. Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy and NEUROFARBA Department, Sezione di Scienze Farmaceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy. † Corresponding Authors: [email protected] (ADF); [email protected] (GDS). Electronic Supplementary Information (ESI) available: Experimental details, Table S1 and Figures S1-S3. See DOI: 10.1039/x0xx00000x b.
Scheme 1 Synthesis of compound 1. The inhibition profile of 1 against all catalytically active hCA isoforms was investigated (Table 1) (see Supplementary information
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for experimental details), showing that this molecule has a rather variable behavior against the different isoforms. Indeed, against hCA II and hCA VA 1 is a potent inhibitor (KIs of 50.9 – 55.5 nM), against several other isoforms (among which hCA IV, IX and XII) it is a medium potency CAI (KIs of 133.6 – 161.9 nM), whereas hCA III, VII and XIII are inhibited with inhibition constants in the 326-608 nM range. Three isoforms (hCA I, VI and XIV) are not inhibited significantly by compound 1. This inhibition profile is unique and of interest to develop inhibitors starting from this lead compound.
additional Raman experiments were performed on the same hCA II native crystals soaked with two other inhibitors, namely 2+ compounds 2 and 3 in Figure 1, whose Zn ion coordination was 12, 13 already defined in previous crystallographic investigations.
Table 1 Inhibition of hCA isozymes I-XIV with compound 1. KI (nM)a hCA I >10000 hCA II 50.9 hCA III 608 b hCA IV 161.9 hCA VA 55.5 hCA VI >10000 hCA VII 325.9 b hCA IX 141.5 hCA XIIb 133.6 hCA XIII 539.1 hCA XIVb >10000 a Errors in the range of ± 5 % of the reported data from three b different assays. Catalytic domain. To elucidate the binding mode of 1 to the CA active site, the structure of the adduct that this molecule forms with the best characterized hCA isoform, namely hCA II, was solved. hCA II/1 crystals were obtained as described in the Supplementary information. The three-dimensional structure was analyzed by difference Fourier techniques. Statistics for data collection and refinement are summarized in Table S1. Inspection of the initially calculated electron density maps in the active site cavity showed clear evidence for the binding of the inhibitor to the catalytic zinc ion. However, these maps, although being very well defined for the protein residues, did not allow to discriminate unambiguously the inhibitor binding mode. Indeed, two different models were compatible with electron density maps: the first one with the catalytic zinc ion coordinated by the sulfonamide nitrogen (hereafter indicated as binding mode A) and the second one where the coordination was due to the 2 11 hydroxylamine moiety in a side-on (η ) fashion (hereafter indicated as binding mode B) (Figure 2). Thus, to get preliminary 2+ information on the Zn coordination in the hCA II/1 complex, Raman microspectroscopy was performed on the hCA II single crystals in the presence and absence of the inhibitor (Figure 3, see Supplementary information for a more detailed description of all Raman experiments). Several Raman signals from hCA II were clearly observed into the crystal (e.g. amide I band around 1666 cm1 -1 , and Phe ring band around 1007 cm ), though in the presence of strong signals from mother liquor. Upon addiction of inhibitor 1, these signals were almost unchanged; however, a relevant Raman band, absent both in the spectra of the native crystals, of the inhibitor and of the mother liquor (Figure 3), appeared around 322 cm-1. This band could be reasonable attributed to a vibrational mode associated to the binding of the inhibitor to the enzyme, in particular, to some stretching modes between Zn2+ and O and/or N atoms. Unfortunately, literature data did not allow an unambiguous assignment of this band (see Supplementary information). Therefore, a crystallography-assisted Raman analysis was adopted:
Figure 2 Active site region in the hCA II/1 adduct, showing the σAweighted |Fo-Fc| OMIT map (contoured at 3.0 σ). The two possible coordination modes of inhibitor 1 are reported. The zinc ion coordination is drawn in black and red for binding mode A and B, respectively. In particular, inhibitor 2 coordinated the catalytic ion through O 12 atoms, while compound 3, the well known CA inhibitor acetazolamide (AZM), bound via the sulfonamide nitrogen atom, 13 exactly in the same way above described for binding mode A. Raman spectra on the hCA II/2 adduct did not show any band -1 around 322 cm , whereas those on the hCA II/3 complex presented -1 a Raman signal around 324 cm , comparable to that observed in the hCA II/1 adduct (Figure 4, Figure S1 and Figure S2). Thus, these crystallography-assisted Raman data suggested that, in analogy 2+ with hCA II/3 complex, at least a partial Zn ion coordination via the sulfonamide nitrogen atom was present in the adduct under investigation, even if other binding coordinations could not be completely ruled out.
Figure 3 Raman spectra of the hCA II native crystals, inhibitor 1 and hCA II/1 complex crystals. Mother liquor (ML) is also reported to detect signals from it (*). Unfortunately, no Raman data were available for side-on Zn2+14, 15 ligand coordination. Thus, we considered some DFT and Raman data corresponding to other metal with nitrosyl or peroxo 15, 16 species. DFT calculations predicted significant structural differences between end-on and side-on coordination,15 while Raman data indicated a significant switch in the low frequency mode between end-on and side-on metal-ligand coordination.17
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Analyzing our Raman spectra we did not observe any additional -1 signal beyond that at 322 cm correspondent to the AZM-like coordination. Thus, we hypothesized that the alternative side-on coordination of the hydroxylamine moiety was not detectable from Raman spectra due to low Raman cross-section. Furthermore, we reasonably assumed that the side-on frequency mode does not fall -1 under the end-on AZM-like Raman band (around 322 cm ), thus allowing the below reported quantitative analysis. Indeed, considering that in the hCA II/3 adduct binding occurs exclusively 13 via N-coordination, our Raman analysis allowed also for a tentatively quantitative evaluation of the AZM-like binding in the hCA II/1 complex. In particular, assuming the Raman cross-section 2+ -1 of the Zn -N band around 322 cm equal for hCA II/1 and hCA II/3 -1 complexes, and using the signal of Phe band at 1007 cm for normalization, a fraction of 0.4 ± 0.1 has been evaluated for AZMlike binding of compound 1 (see Figure S3). Thus, altogether Raman 2+ data suggest that the coordination of the catalytic Zn ion in the hCA II/1 complex occurs for the 40% through the sulfonamide nitrogen (binding conformation A) and for the remaining 60% through a binding coordination not identifiable from Raman experiments.
Figure 4 Raman spectra comparison among wild type hCA II and three complexes with the corresponding compounds. On the basis of these data we modeled the two, initially hypothesized, alternative binding conformations in the electron density maps (Figure 2), and, after fixing their B-factor to values comparable to those of the solvent within the active site, we refined their occupancy factors. Interestingly, in good agreement with Raman spectroscopy data, we found Q=0.33 for binding conformation A, and Q=0.34 for binding conformation B. At this point, since the total occupancy of the inhibitor in both conformations was 0.67 and residual difference electron density was still present close to the zinc ion, we added in our model a water molecule with an occupancy factor Q=0.33, coordinated to the zinc ion and alternative to ligand binding. The main proteininhibitor interactions for both binding modes A and B are shown in Figure 5. Interestingly, even if many studies suggest that sulfonamides generally coordinate to the catalytic zinc ion through a deprotonated nitrogen atom, the pKa values calculated for the hydroxylamine and sulfonamide moieties of compound 1 by the the Marvin software developed by ChemAxon (www.chemaxon.com/marvin/sketch/index.php), suggest that at the pH used for crystallization experiments, in both binding modes
the inhibitor is coordinated to the metal ion in a not deprotonated form. 18 It is also worth of note that compound 4, a strictly related isomer of compound 1 (Figure 1), adopted only one conformation within the hCA II active site, coordinating the zinc ion through the sulfonamide nitrogen atom (Figure 6A and 6B), as observed in conformation A of the hCA II/1 adduct.
2+
Figure 5 Zn coordination geometry in the hCA II/1 adduct for binding conformation A (A) and B (B). Hydrogen bonds and residues forming van der Waals interactions are also reported.
2+
Figure 6 (A) Zn coordination geometry in the hCA II/4 adduct (PDB 18 code 2O4Z). (B) Structural superposition of 1 in conformation A and 4 when bound to the hCA II active site. The active site residues of hCA II/1 and hCA II/4 adducts are colored in pink and green, respectively. Inhibitor polar interactions are drawn in black for hCA II/1 and red for hCA II/4. We also compared the inhibitor binding mode observed in hCA II/1 19 with that of a classical benzene-sulfonamide, namely compound 5 (Figure 7A and 7B). Interestingly, while the sulfonamide moiety of 1 in the binding conformation B is perfectly superimposable to the sulfonamide group of 5, the same moiety in binding conformation A assumes a position completely different from that of benzenesulfonamide 5. On the basis of these data, it is possible to hypothesize that derivatization of compound 1 on the hydroxylamine moiety, thus forcing the binding conformation A, could allow the inhibitor to interact with regions of the active site not yet explored with classical benzene-sulfonamides. Altogether, these results strongly suggest that hydroxylamine-O-sulfonamide inhibitor represents an interesting lead compound alternative to the classical sulfonamides for the development of more selective CAIs.
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The last years saw the discovery of an entire new range of CAIs 1 possessing a variety of non-classical inhibition mechanisms. Indeed, for more than 60 years, since the report of sulfonamides as CAIs, the drug design landscape was dominated by these compounds and their isosteres, the sulfamates and sulfamides.
Figure 7. Structural superposition of inhibitor 5 with inhibitor 1 in conformation B (A) and in conformation A (B) when bound to the hCA II active site. The inhibitor 5 is colored in magenta. They possess a rather simple inhibition mechanism, as they bind in deprotonated form to the metal ion from the active site, as the 1 fourth ligand. However, starting with 2008 completely new chemotypes were reported as CAIs: the coumarins,20 the 21 22 23 12 polyamines, dithiocarbamates, xanthates, hydroxamate, sulfocoumarins,24 carboxylates, etc.10, 25 Many of them possess inhibition mechanism strikingly different compared to the classical one. Indeed, some of these inhibitors anchor to the zinccoordinated water molecule (polyamines, sulfocoumarins, some carboxylates),21, 24, 25 others bind towards the exit of the active site cavity in the activator-binding site (coumarins and their derivatives),20, 26 whereas some carboxylates are even observed 10 bound out-of-the active site. The compound investigated here, hydroxylamine-O-sulfonamide 1, although possessing a simple chemical structure, is unique due to its versatility in inhibiting these enzymes, being the first example in which two alternative coordinations to the metal ion were evidenced. Indeed, as shown above, compound 1 is in part coordinated in the classical manner, 2+ as all sulfonamides/sulfamates, binding the Zn ion through the sulfonamide nitrogen and in part through hydroxylamine moiety in 2 a side-on (η ) fashion, which is an unusual inhibition pattern for this family of enzymes. This versatility can definitely be exploited for drug design purposes since the chemical simplicity of the compound 1 is amenable to elaboration through medicinal chemistry purposes. Work is in progress in our laboratories for designing CAIs incorporating this new lead compound, which may lead to more selective/potent inhibitors for the various isoforms with biomedical applications. It is also worth noting that in this paper an original crystallography-assisted Raman spectroscopy approach was utilized to investigate on the binding mechanism of 27,28 compound 1 to hCA II. This approach allowed to identify the 2+ hydroxylamine-O-sulfonamide binding mode to Zn and demonstrated to have a more general utility in the investigation of metal ion coordination when crystallographic or Raman data alone cannot provide an unambiguous response.
Notes and references
§ This work was supported by a grant by CNR-DSB. Progetto Bandiera "InterOmics”. AV acknowledges "POR BIP (Bioindustrial Industrial Processes)" for a research grant 1. V. Alterio, A. Di Fiore, K. D'Ambrosio, C. T. Supuran and G. De Simone, Chem. Rev., 2012, 112, 4421. 2. C. T. Supuran, Nat. Rev. Drug Discov., 2008, 7, 168. 3. G. De Simone, A. Di Fiore, C. Capasso and C. T. Supuran, Bioorg. Med. Chem. Lett., 2015, 25, 1385. 4. C. T. Supuran, A. Di Fiore and G. De Simone, Expert Opin. Emerg. Drugs, 2008, 13, 383. 5. C. T. Supuran, A. Di Fiore, V. Alterio, S. M. Monti and G. De Simone, Curr. Pharm. Des., 2010, 16, 3246. 6. R. Del Giudice, D. M. Monti, E. Truppo, A. Arciello, C. T. Supuran, G. De Simone and S. M. Monti, Biol. Chem., 2013, 394, 1343. 7. Carbonic Anhydrases as Biocatalysts - From Theory to Medical and Industrial Applications, Supuran, C. T. and De Simone, G. eds., Elsevier B. V., 2015. 8. G. De Simone, A. Scozzafava and C. T. Supuran, Chem. Biol. Drug. Des., 2009, 74, 317. 9. J.-Y. Winum and C. T. Supuran, J. Enzyme Inhib. Med. Chem., 2015, 30, 321. 10. K. D'Ambrosio, S. Carradori, S. M. Monti, M. Buonanno, D. Secci, D. Vullo, C. T. Supuran and G. De Simone, Chem. Commun., 2015, 51, 302. 11. C. W. Belock, A. C¸etin, N. V. Barone and C. J. Ziegler, Inorg. Chem., 2008, 47, 7114. 12. A. Di Fiore, A. Maresca, C. T. Supuran and G. De Simone, Chem. Commun., 2012, 48, 8838. 13. K. H. Sippel, A. H. Robbins, J. Domsic, C. Genis, M. AgbandjeMcKenna and R. McKenna, Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun., 2009, 65, 992. 14. C. De La Cruz and N. Sheppard, Spectrochim. Acta, Pt. A: Mol. Spectrosc., 2011, 78, 7. 15. H. Chen, K.-B. Cho, W. Lai, W. Nam and S. Shaik, J. Chem. Theory Comput., 2012, 8, 915−926. 16. J. Cho, R. Sarangi and W. Nam, Acc. Chem. Res., 2012, 45, 1321. 17. J. G. Liu, T. Ohta, S. Yamaguchi, T. Ogura, S. Sakamoto, Y. Maeda and Y. Naruta, Angew. Chem., 2009, 48, 9262. 18. C. Temperini, J. Y. Winum, J. L. Montero, A. Scozzafava and C. T. Supuran, Bioorg. Med. Chem. Lett., 2007, 17, 2795. 19. A. D. Scott, C. Phillips, A. Alex, M. Flocco, A. Bent, A. Randall, R. O'Brien, L. Damian and L. H. Jones, ChemMedChem, 2009, 4, 1985. 20. A. Maresca, C. Temperini, H. Vu, N. B. Pham, S. A. Poulsen, A. Scozzafava, R. J. Quinn and C. T. Supuran, J. Am. Chem. Soc., 2009, 131, 3057. 21. F. Carta, C. Temperini, A. Innocenti, A. Scozzafava, K. Kaila and C. T. Supuran, J. Med. Chem., 2010, 53, 5511. 22. F. Carta, M. Aggarwal, A. Maresca, A. Scozzafava, R. McKenna and C. T. Supuran, Chem. Commun., 2012, 48, 1868. 23. F. Carta, A. Akdemir, A. Scozzafava, E. Masini and C. T. Supuran, J. Med. Chem., 2013, 56, 4691. 24. K. Tars, D. Vullo, A. Kazaks, J. Leitans, A. Lends, A. Grandane, R. Zalubovskis, A. Scozzafava and C. T. Supuran, J. Med. Chem., 2013, 56, 293. 25. D. P. Martin and S. M. Cohen, Chem. Commun., 2012, 48, 5259. 26. A. Maresca, C. Temperini, L. Pochet, B. Masereel, A. Scozzafava and C. T. Supuran, J. Med. Chem., 2010, 53, 335. 27. A. Vergara, G. D'Errico, D. Montesarchio, G. Mangiapia, L. Paduano and A. Merlino, Inorg. Chem., 2013, 52, 4157. 28. A. Vergara, L. Vitagliano, A. Merlino, F. Sica, K. Marino, C. Verde, G. di Prisco and L. Mazzarella, J. Biol. Chem., 2010, 285, 32568.
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Received 00th January 20xx, Accepted 00th January 20xx DOI: 10.1039/x0xx00000x www.rsc.org/
HYDROXYLAMINE-O-SULFONAMIDE IS A VERSATILE LEAD COMPOUND FOR THE DEVELOPMENT OF CARBONIC ANHYDRASE INHIBITORS Anna Di Fiore,a† Alessandro Vergara,a,b Marco Caterino,b Vincenzo Alterio,a Simona M. Monti,a Joanna Ombouma,c Pascal Dumy,c Daniela Vullo,d Claudiu T. Supuran,d Jean-Yves Winum,c and Giuseppina De Simonea†
Hydroxylamine-O-sulfonamide, a molecule incorporating two zincbinding groups (ZBGs), has been investigated as carbonic anhydrase inhibitor (CAI) by means of kinetic, crystallographic and Raman spectroscopy studies, highlighting interesting results on its mechanism of action. These data can be exploited to design new effective and selective CAIs. Carbonic anhydrases (CAs) are ubiquitous metallo-enzymes which 1, 2 catalyze the reversible CO2 hydration reaction. These enzymes are widespread in all the kingdoms of life and are encoded by six evolutionarily unrelated gene families: α-, β-, γ-, δ-, ζ- and η-CAs.1, 3 Human (h) CAs belong to the α-class and are present in 15 isoforms, which differ by molecular features, oligomeric arrangement, cellular 1 localization, tissue distribution, and kinetic properties. Considering the crucial role of the CA catalyzed reaction, these enzymes are 4-8 involved in a variety of physiological/pathological processes, and consequently have lately become important targets for 1 pharmaceutical research. In recent years, numerous studies have been reported on CA inhibitors (CAIs). In particular, the elucidation of their mechanism of action, through the crystallographic analysis of several enzymeinhibitor adducts, allowed to determine the molecular basis of enzyme-inhibitor interactions.1 The most effective CAIs are sulfonamide/sulfamate-based molecules that interact with the enzyme active site coordinating the zinc ion and displacing the 1 water molecule/hydroxide ion present in the native enzyme; however, these molecules are generally poorly selective towards the different CA isoforms. For this reason, despite many encouraging results, new zinc binding groups (ZBGs) are continuously tested in order to advance upon the identification of
9
isoform selective CAIs.
In this paper, we report the synthesis, inhibition and structural studies of hydroxylamine-O-sulfonamide 1 (Figure 1), a molecule containing two ZBGs, namely the sulfonamide and hydroxylamine moieties. Our initial working hypothesis was that the concurrent presence of two functional groups could give an ambidentate character to this derivative. Our data show that compound 1 possesses an interesting inhibition profile against the different hCA isoforms as well as an intriguing binding mode. Indeed, it can bind the zinc ion of the CA active site with both ZBGs, thus suggesting that the derivatization both of the sulfonamide nitrogen and of the hydroxylamine moiety can be used to obtain new classes of 10 inhibitors.
Figure 1 Chemical structures of compounds 1–5. Compound 1 was synthesized in a two step synthesis starting from the commercially available N-Boc-hydroxylamine A as depicted in Scheme 1. Reaction of sulfamoylation of A with sulfamoyl chloride led quantitatively to the N-Boc-O-sulfamoyl hydroxylamine B. The latter was then reacted with a solution of TFA in dichloromethane to afford the hydroxylamine-O-sulfonamide 1 as trifluoroacetate salt (see Scheme 1 and Supplementary information for details).
a.
Istituto di Biostrutture e Bioimmagini-CNR, via Mezzocannone 16, 80134 Napoli, Italy. Department of Chemical Sciences, University of Naples Federico II, Napoli, Via Cinthia 80126, Italy. c. Institut des Biomolécules Max Mousseron (IBMM) UMR 5247 CNRS, ENSCM, Université de Montpellier, Bâtiment de Recherche Max Mousseron, Ecole Nationale Supérieure de Chimie de Montpellier, 8 rue de l’Ecole Normale, 34296 Montpellier Cedex, France. d. Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy and NEUROFARBA Department, Sezione di Scienze Farmaceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy. † Corresponding Authors: [email protected] (ADF); [email protected] (GDS). Electronic Supplementary Information (ESI) available: Experimental details, Table S1 and Figures S1-S3. See DOI: 10.1039/x0xx00000x b.
Scheme 1 Synthesis of compound 1. The inhibition profile of 1 against all catalytically active hCA isoforms was investigated (Table 1) (see Supplementary information
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for experimental details), showing that this molecule has a rather variable behavior against the different isoforms. Indeed, against hCA II and hCA VA 1 is a potent inhibitor (KIs of 50.9 – 55.5 nM), against several other isoforms (among which hCA IV, IX and XII) it is a medium potency CAI (KIs of 133.6 – 161.9 nM), whereas hCA III, VII and XIII are inhibited with inhibition constants in the 326-608 nM range. Three isoforms (hCA I, VI and XIV) are not inhibited significantly by compound 1. This inhibition profile is unique and of interest to develop inhibitors starting from this lead compound.
additional Raman experiments were performed on the same hCA II native crystals soaked with two other inhibitors, namely 2+ compounds 2 and 3 in Figure 1, whose Zn ion coordination was 12, 13 already defined in previous crystallographic investigations.
Table 1 Inhibition of hCA isozymes I-XIV with compound 1. KI (nM)a hCA I >10000 hCA II 50.9 hCA III 608 b hCA IV 161.9 hCA VA 55.5 hCA VI >10000 hCA VII 325.9 b hCA IX 141.5 hCA XIIb 133.6 hCA XIII 539.1 hCA XIVb >10000 a Errors in the range of ± 5 % of the reported data from three b different assays. Catalytic domain. To elucidate the binding mode of 1 to the CA active site, the structure of the adduct that this molecule forms with the best characterized hCA isoform, namely hCA II, was solved. hCA II/1 crystals were obtained as described in the Supplementary information. The three-dimensional structure was analyzed by difference Fourier techniques. Statistics for data collection and refinement are summarized in Table S1. Inspection of the initially calculated electron density maps in the active site cavity showed clear evidence for the binding of the inhibitor to the catalytic zinc ion. However, these maps, although being very well defined for the protein residues, did not allow to discriminate unambiguously the inhibitor binding mode. Indeed, two different models were compatible with electron density maps: the first one with the catalytic zinc ion coordinated by the sulfonamide nitrogen (hereafter indicated as binding mode A) and the second one where the coordination was due to the 2 11 hydroxylamine moiety in a side-on (η ) fashion (hereafter indicated as binding mode B) (Figure 2). Thus, to get preliminary 2+ information on the Zn coordination in the hCA II/1 complex, Raman microspectroscopy was performed on the hCA II single crystals in the presence and absence of the inhibitor (Figure 3, see Supplementary information for a more detailed description of all Raman experiments). Several Raman signals from hCA II were clearly observed into the crystal (e.g. amide I band around 1666 cm1 -1 , and Phe ring band around 1007 cm ), though in the presence of strong signals from mother liquor. Upon addiction of inhibitor 1, these signals were almost unchanged; however, a relevant Raman band, absent both in the spectra of the native crystals, of the inhibitor and of the mother liquor (Figure 3), appeared around 322 cm-1. This band could be reasonable attributed to a vibrational mode associated to the binding of the inhibitor to the enzyme, in particular, to some stretching modes between Zn2+ and O and/or N atoms. Unfortunately, literature data did not allow an unambiguous assignment of this band (see Supplementary information). Therefore, a crystallography-assisted Raman analysis was adopted:
Figure 2 Active site region in the hCA II/1 adduct, showing the σAweighted |Fo-Fc| OMIT map (contoured at 3.0 σ). The two possible coordination modes of inhibitor 1 are reported. The zinc ion coordination is drawn in black and red for binding mode A and B, respectively. In particular, inhibitor 2 coordinated the catalytic ion through O 12 atoms, while compound 3, the well known CA inhibitor acetazolamide (AZM), bound via the sulfonamide nitrogen atom, 13 exactly in the same way above described for binding mode A. Raman spectra on the hCA II/2 adduct did not show any band -1 around 322 cm , whereas those on the hCA II/3 complex presented -1 a Raman signal around 324 cm , comparable to that observed in the hCA II/1 adduct (Figure 4, Figure S1 and Figure S2). Thus, these crystallography-assisted Raman data suggested that, in analogy 2+ with hCA II/3 complex, at least a partial Zn ion coordination via the sulfonamide nitrogen atom was present in the adduct under investigation, even if other binding coordinations could not be completely ruled out.
Figure 3 Raman spectra of the hCA II native crystals, inhibitor 1 and hCA II/1 complex crystals. Mother liquor (ML) is also reported to detect signals from it (*). Unfortunately, no Raman data were available for side-on Zn2+14, 15 ligand coordination. Thus, we considered some DFT and Raman data corresponding to other metal with nitrosyl or peroxo 15, 16 species. DFT calculations predicted significant structural differences between end-on and side-on coordination,15 while Raman data indicated a significant switch in the low frequency mode between end-on and side-on metal-ligand coordination.17
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Analyzing our Raman spectra we did not observe any additional -1 signal beyond that at 322 cm correspondent to the AZM-like coordination. Thus, we hypothesized that the alternative side-on coordination of the hydroxylamine moiety was not detectable from Raman spectra due to low Raman cross-section. Furthermore, we reasonably assumed that the side-on frequency mode does not fall -1 under the end-on AZM-like Raman band (around 322 cm ), thus allowing the below reported quantitative analysis. Indeed, considering that in the hCA II/3 adduct binding occurs exclusively 13 via N-coordination, our Raman analysis allowed also for a tentatively quantitative evaluation of the AZM-like binding in the hCA II/1 complex. In particular, assuming the Raman cross-section 2+ -1 of the Zn -N band around 322 cm equal for hCA II/1 and hCA II/3 -1 complexes, and using the signal of Phe band at 1007 cm for normalization, a fraction of 0.4 ± 0.1 has been evaluated for AZMlike binding of compound 1 (see Figure S3). Thus, altogether Raman 2+ data suggest that the coordination of the catalytic Zn ion in the hCA II/1 complex occurs for the 40% through the sulfonamide nitrogen (binding conformation A) and for the remaining 60% through a binding coordination not identifiable from Raman experiments.
Figure 4 Raman spectra comparison among wild type hCA II and three complexes with the corresponding compounds. On the basis of these data we modeled the two, initially hypothesized, alternative binding conformations in the electron density maps (Figure 2), and, after fixing their B-factor to values comparable to those of the solvent within the active site, we refined their occupancy factors. Interestingly, in good agreement with Raman spectroscopy data, we found Q=0.33 for binding conformation A, and Q=0.34 for binding conformation B. At this point, since the total occupancy of the inhibitor in both conformations was 0.67 and residual difference electron density was still present close to the zinc ion, we added in our model a water molecule with an occupancy factor Q=0.33, coordinated to the zinc ion and alternative to ligand binding. The main proteininhibitor interactions for both binding modes A and B are shown in Figure 5. Interestingly, even if many studies suggest that sulfonamides generally coordinate to the catalytic zinc ion through a deprotonated nitrogen atom, the pKa values calculated for the hydroxylamine and sulfonamide moieties of compound 1 by the the Marvin software developed by ChemAxon (www.chemaxon.com/marvin/sketch/index.php), suggest that at the pH used for crystallization experiments, in both binding modes
the inhibitor is coordinated to the metal ion in a not deprotonated form. 18 It is also worth of note that compound 4, a strictly related isomer of compound 1 (Figure 1), adopted only one conformation within the hCA II active site, coordinating the zinc ion through the sulfonamide nitrogen atom (Figure 6A and 6B), as observed in conformation A of the hCA II/1 adduct.
2+
Figure 5 Zn coordination geometry in the hCA II/1 adduct for binding conformation A (A) and B (B). Hydrogen bonds and residues forming van der Waals interactions are also reported.
2+
Figure 6 (A) Zn coordination geometry in the hCA II/4 adduct (PDB 18 code 2O4Z). (B) Structural superposition of 1 in conformation A and 4 when bound to the hCA II active site. The active site residues of hCA II/1 and hCA II/4 adducts are colored in pink and green, respectively. Inhibitor polar interactions are drawn in black for hCA II/1 and red for hCA II/4. We also compared the inhibitor binding mode observed in hCA II/1 19 with that of a classical benzene-sulfonamide, namely compound 5 (Figure 7A and 7B). Interestingly, while the sulfonamide moiety of 1 in the binding conformation B is perfectly superimposable to the sulfonamide group of 5, the same moiety in binding conformation A assumes a position completely different from that of benzenesulfonamide 5. On the basis of these data, it is possible to hypothesize that derivatization of compound 1 on the hydroxylamine moiety, thus forcing the binding conformation A, could allow the inhibitor to interact with regions of the active site not yet explored with classical benzene-sulfonamides. Altogether, these results strongly suggest that hydroxylamine-O-sulfonamide inhibitor represents an interesting lead compound alternative to the classical sulfonamides for the development of more selective CAIs.
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The last years saw the discovery of an entire new range of CAIs 1 possessing a variety of non-classical inhibition mechanisms. Indeed, for more than 60 years, since the report of sulfonamides as CAIs, the drug design landscape was dominated by these compounds and their isosteres, the sulfamates and sulfamides.
Figure 7. Structural superposition of inhibitor 5 with inhibitor 1 in conformation B (A) and in conformation A (B) when bound to the hCA II active site. The inhibitor 5 is colored in magenta. They possess a rather simple inhibition mechanism, as they bind in deprotonated form to the metal ion from the active site, as the 1 fourth ligand. However, starting with 2008 completely new chemotypes were reported as CAIs: the coumarins,20 the 21 22 23 12 polyamines, dithiocarbamates, xanthates, hydroxamate, sulfocoumarins,24 carboxylates, etc.10, 25 Many of them possess inhibition mechanism strikingly different compared to the classical one. Indeed, some of these inhibitors anchor to the zinccoordinated water molecule (polyamines, sulfocoumarins, some carboxylates),21, 24, 25 others bind towards the exit of the active site cavity in the activator-binding site (coumarins and their derivatives),20, 26 whereas some carboxylates are even observed 10 bound out-of-the active site. The compound investigated here, hydroxylamine-O-sulfonamide 1, although possessing a simple chemical structure, is unique due to its versatility in inhibiting these enzymes, being the first example in which two alternative coordinations to the metal ion were evidenced. Indeed, as shown above, compound 1 is in part coordinated in the classical manner, 2+ as all sulfonamides/sulfamates, binding the Zn ion through the sulfonamide nitrogen and in part through hydroxylamine moiety in 2 a side-on (η ) fashion, which is an unusual inhibition pattern for this family of enzymes. This versatility can definitely be exploited for drug design purposes since the chemical simplicity of the compound 1 is amenable to elaboration through medicinal chemistry purposes. Work is in progress in our laboratories for designing CAIs incorporating this new lead compound, which may lead to more selective/potent inhibitors for the various isoforms with biomedical applications. It is also worth noting that in this paper an original crystallography-assisted Raman spectroscopy approach was utilized to investigate on the binding mechanism of 27,28 compound 1 to hCA II. This approach allowed to identify the 2+ hydroxylamine-O-sulfonamide binding mode to Zn and demonstrated to have a more general utility in the investigation of metal ion coordination when crystallographic or Raman data alone cannot provide an unambiguous response.
Notes and references
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