Carbonic anhydrase inhibitors: X-ray crystallographic studies for the binding of 5-amino-1,3,4-thiadiazole-2-sulfonamide and 5-(4-amino-3-chloro-5-fluorophenylsulfonamido)- 1,3,4-thiadiazole-2-sulfonamide to human isoform II Valeria Menchise, a Giuseppina De Simone, a, * Anna Di Fiore, a Andrea Scozzafava b and Claudiu T. Supuran b, * a Istituto di Biostrutture e Bioimmagini-CNR, via Mezzocannone 16, 80134 Naples, Italy b Universita ` degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, I-50019 Sesto Fiorentino (Firenze), Italy Received 18 July 2006; revised 7 September 2006; accepted 9 September 2006 Available online 26 September 2006 Abstract—The X-ray crystal structures of 5-amino-1,3,4-thiadiazole-2-sulfonamide (the acetazolamide precursor) and 5-(4-amino-3- chloro-5-fluorophenylsulfonamido)-1,3,4-thiadiazole-2-sulfonamide in complex with the human isozyme II of carbonic anhydrase (CA, EC 4.2.1.1) are reported. The thiadiazole-sulfonamide moiety of the two compounds binds in the canonic manner to the zinc ion and interacts with Thr199, Glu106, and Thr200. The substituted phenyl tail of the second inhibitor was positioned in the hydro- phobic part of the binding pocket, at van der Waals distance from Phe131, Val 135, Val141, Leu198, Pro202, and Leu204. These structures may help in the design of better inhibitors of these widespread zinc-containing enzymes. Ó 2006 Elsevier Ltd. All rights reserved. The metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) catalyzes a very simple but critically important physiolog- ical reaction: the interconversion between carbon diox- ide, generated in huge amounts in all metabolic processes, and the bicarbonate ion. 1–6 Inhibitors of these zinc-containing enzymes show a multitude of applica- tions as diuretic, antiglaucoma, antiobesity or antitumor drugs, being also used as diagnostic tools. 1–6 Various CA isoforms are responsible for specific physiological func- tions, and drugs with such a diversity of actions target dif- ferent isozymes of the 15 presently known in humans. 2–6 In all of them, the inhibitor is bound as anion to the cat- alytically critical Zn 2+ ion, also participating in extensive hydrogen bond networks and van der Waals interactions with amino acid residues both in the hydrophobic and hydrophilic halves of the enzyme active site, as shown by X-ray crystallographic studies of such enzyme–inhibi- tor complexes. 7–14 Among the three main classes of po- tent CA inhibitors (CAIs) described up to now, the sulfonamides, the sulfamates, and the sulfamides, the first one is the most investigated, since classical, clinically used drugs such as acetazolamide 1, methazolamide 2, ethox- zolamide 3, dichlorophenamide 4, dorzolamide 5, and brinzolamide 6 all belong to it. 1–6 X-ray crystal stuctures are available for adducts of several isozymes (i.e., CA I, II, IV, V, XII, and XIV) 7–14 mostly with sulfonamides, with several sulfamates and few sulfamides. 10 Novel sulfonamide derivatives are thus constantly syn- thesized and investigated as inhibitors of various CA en- zymes (from vertebrates or other organisms) in the search of compounds with selectivity for some of the many physiologically relevant isoforms, or for new applications, since the clinically used drugs 1–6 unselec- tively inhibit many CA isozymes and as a consequence, show many undesired side effects. 1–6 For example, a fluorescent sulfonamide recently reported by our group is in clinical development as a diagnostic agent, allowing the imaging of acute hypoxic tumors which are non-re- sponsive to classical chemo- and radio-therapy. 7,15 0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2006.09.022 Keywords: Carbonic anhydrase; Sulfonamide; 1,3,4-Thiadiazole; X-ray crystallography; Enzyme inhibitor. * Corresponding authors. Tel.: +39 081 2534579; fax: +39 081 2536642 (G.D.S.), tel.: +39 055 4573005; fax: +39 055 4573385 (C.T.S.); e-mail addresses: gdesimon@unina.it; claudiu.supuran@unifi.it Bioorganic & Medicinal Chemistry Letters 16 (2006) 6204–6208