A new b-carbonic anhydrase from Brucella suis, its cloning, characterization, and inhibition with sulfonamides and sulfamates, leading to impaired pathogen growth Pascale Joseph a , Safia Ouahrani-Bettache a , Jean-Louis Montero b , Isao Nishimori c , Tomoko Minakuchi c , Daniela Vullo d , Andrea Scozzafava d , Jean-Yves Winum b,⇑ , Stephan Köhler a,⇑ , Claudiu T. Supuran d,⇑ a Centre d’Etudes d’Agents Pathogènes et Biotechnologies pour la Santé (CPBS), UMR 5236 CNRS-UM1-UM2, Université Montpellier II, cc100, Place E. Bataillon, 34095 Montpellier, France b Institut des Biomolécules Max Mousseron (IBMM), UMR 5247 CNRS-UM1-UM2, Bâtiment de Recherche Max Mousseron, Ecole Nationale Supérieure de Chimie de Montpellier, 8 rue de l’Ecole Normale, 34296 Montpellier Cedex, France c Department of Gastroenterology, Kochi Medical School, Nankoku, Kochi 783-8505, Japan d Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy article info Article history: Received 2 September 2010 Revised 22 December 2010 Accepted 23 December 2010 Available online 30 December 2010 Keywords: Carbonic anhydrase b-Class enzyme Brucella suis Sulfonamide Sulfamate Antibacterials abstract A b-carbonic anhydrase (CA, EC 4.2.1.1) from the bacterial pathogen Brucella suis, bsCA II, has been cloned, purified, and characterized kinetically. bsCA II showed high catalytic activity for the hydration of CO 2 to bicarbonate, with a k cat of 1.1 Â 10 6 , and k cat /K m of 8.9 Â 10 7 M À1 s À1 . A panel of sulfonamides and sul- famates have been investigated for inhibition of this enzyme. All types of activities, from the low nanomolar to the micromolar, have been detected for these derivatives, which showed inhibition constants in the range of 7.3 nM–8.56 lM. The best bsCA II inhibitors were some glycosylated sulfanila- mides, aliphatic sulfamates, and halogenated sulfanilamides, with inhibition constants of 7.3–87 nM. Some of these dual inhibitors of bsCA I and II, also inhibited bacterial growth in vitro, in liquid cultures. These promising data on live bacteria allow us to propose bacterial b-CA inhibition as an approach for obtaining anti-infective agents with a new mechanism of action compared to classical antibiotics. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Bacterial infections constitute an alarming health problem worldwide. The widespread emergence of resistance and multi- resistance to antibiotics among bacterial pathogens represents a major threat and necessitates a permanent race for new molecules. The challenge at present is to identify and to validate novel phar- maceutical targets in bacteria, starting point for the discovery of new classes of antibacterial agents that could circumvent the established mechanisms of resistance. 1 Among the many antibacterial drug targets available so far from bacterial genomics studies, metalloenzymes are highly attractive as they provide an excellent opportunity for mechanism-based drug discovery of novel classes of antibiotics. 2 In this framework, carbonic anhydrases (CAs, EC 4.2.1.1) have recently emerged as promising anti-infective targets. 3,4 Indeed, several bacterial b-CA class representatives have been cloned and characterized in some pathogens such as, among others, Helicobacter pylori and Mycobac- terium tuberculosis. 5,6 Inhibition or genetic silencing studies of the b-CA from H. pylori proved it to be critical for the growth and vir- ulence of this pathogen. 5,7 Thus, inhibition of b-CAs 8 can be consid- ered as a new possible approach for designing antibacterial agents possessing a different mechanism of action than classical pharma- cological agents in clinical use for a long period, for which many pathogenic bacteria developed various degrees of resistance. 3,4 By searching for metalloenzyme-specific sequence motifs with- in the Brucella suis genome 9 (the pathogen responsible of brucello- sis, the most widespread bacterial zoonosis worldwide), 10 we identified two genes encoding carbonic anhydrases belonging to the b-CA superfamily 8 (BR1829 and BRA0788). Indeed, these enzymes are widespread in organisms all over the phylogenetic tree, with five different families encoding them, the a-, b-, c-, d-, and f-CAs. 2–4 All of them are metalloenzymes, but whereas a-, b-, and d-CAs use Zn(II) ions at the active site, 2–4 the c-CAs are probably Fe(II) enzymes (but they are active also with bound Zn(II) or Co(II) ions), whereas the f-class uses Cd(II) or Zn(II) to perform the physiologic reaction catalysis. 2–4 The 3D folds of the five enzyme classes are very different from each other, as is their 0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2010.12.048 ⇑ Corresponding authors. Tel.: +39 055 4573005; fax: +39 055 4573385 (C.T.S.). E-mail addresses: jean-yves.winum@univ-montp2.fr (J.-Y. Winum), stephan.- kohler@cpbs.cnrs.fr (S. Köhler), claudiu.supuran@unifi.it (C.T. Supuran). Bioorganic & Medicinal Chemistry 19 (2011) 1172–1178 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc