CAs 4423 3. Insights into CA Catalytic Mechanism: CO 2 and HCO 3 − Binding to hCA II 4423 4. Insights into CA Inhibition: Structural Features of Zinc Binding Inhibitors 4424 4.1. Binding of Ureates and Hydroxamates 4425 4.2. Thiol Derivatives 4426 4.3. Metal-Complexing Anions 4428 4.4. Sulfonamides 4428 4.4.1. Benzenesulfonamides 4428 4.4.2. Thiophene, Thiadiazole, and Thiadiazoline Derivatives 4440 4.4.3. Sulfonamides Containing Other Ring Systems 4443 4.4.4. Thiazide Diuretics 4445 4.4.5. Aliphatic Sulfonamides 4446 4.5. Sulfamates and Sulfamides 4447 4.5.1. Aliphatic Sulfamates 4448 4.5.2. Sulfamate CAIs Also Acting as Steroid Sulfatase and Aromatase Inhibitors 4450 4.6. Sulfonamides/Sulfamates/Sulfamides Containing Sugar Moieties 4452 5. Insights into CA Inhibition: Structural Features of Non-Zinc-Binding Inhibitors 4455 5.1. Compounds Anchoring to the Zinc Bound Water Molecule 4455 5.1.1. Phenols 4455 5.1.2. Spermine and Related Polyamines 4456 5.2. Compounds Located at the Entrance of the Active Site: Coumarins and Lacosamide 4457 6. Conclusions 4459 Author Information 4461 Corresponding Author 4461 Notes 4461 Biographies 4461 Acknowledgments 4462 Abbreviations 4462 References 4462

Carbonic anhydrase (CA) IX is a plasma membrane-associated member of the ␣-CA enzyme family, which is involved in solid tumor acidification. It is a marker of tumor hypoxia and a prognostic factor in several human cancers. An aberrant increase in CA IX expression in chronic hypoxia and during development of various carcinomas contributes to tumorigenesis through at least two mechanisms: pH regulation and cell adhesion control. Here we report the X-ray structure of the catalytic domain of CA IX in complex with a classical, clinically used sulfonamide inhibitor, acetazolamide. The structure reveals a typical ␣-CA fold, which significantly differs from the other CA isozymes when the protein quaternary structure is considered. Thus, two catalytic domains of CA IX associate to form a dimer, which is stabilized by the formation of an intermolecular disulfide bond. The active site clefts and the PG domains are located on one face of the dimer, while the C-termini are located on the opposite face to facilitate protein anchoring to the cell membrane. A correlation between the threedimensional structure and the physiological role of the enzyme is here suggested, based on the measurement of the pH profile of the catalytic activity for the physiological reaction, CO 2 hydration to bicarbonate and protons. On the basis of the structural differences observed between CA IX and the other membrane-associated ␣-CAs, further prospects for the rational drug design of isozymespecific CA inhibitors are proposed, given that inhibition of this enzyme shows antitumor activity both in vitro and in vivo.

Although amazing progress has been made in the structure-based drug design of CAIs, this field is still in progress, with many constantly emerging new findings. Indeed, several new such enzymes were discovered and characterized recently and novel chemotypes were explored for finding compounds with a better inhibition profile. It is anticipated that this will continue to be one of the main frontiers in the search of pharmacologically relevant enzyme inhibitors.

Human carbonic anhydrase (CA) IX is a tumor-associated protein, since it is scarcely present in normal tissues, but highly overexpressed in a large number of solid tumors, where it actively contributes to survival and metastatic spread of tumor cells. Due to these features, the characterization of its biochemical, structural, and functional features for drug design purposes has been extensively carried out, with consequent development of several highly selective small molecule inhibitors and monoclonal antibodies to be used for different purposes. Aim of this review is to provide a comprehensive state-of-the-art of studies performed on this enzyme, regarding structural, functional, and biomedical aspects, as well as the development of molecules with diagnostic and therapeutic applications for cancer treatment. A brief description of additional pharmacologic applications for CA IX inhibition in other diseases, such as arthritis and ischemia, is also provided.

The X-ray crystal structure of the fluorescent antitumor sulfonamide carbonic anhydrase (CA, EC, 4.2.1.1) inhibitor (4-sulfamoylphenylethyl)thioureido fluorescein (1) in complex with the cytosolic isoform hCA II is reported, together with a modeling study of the adduct of 1 with the tumor-associated isoform hCA IX. Its binding to hCA II is similar to that of other benzesulfonamides, with the ionized sulfonamide coordinated to the Zn2+ ion within the enzyme active site, and also participating in a network of hydrogen bonds with residues Thr199 and Glu106. The scaffold of 1 did not establish polar interactions within the enzyme active site but made hydrophobic contacts (<4.5 A) with Gln92, Val121, Phe131, Val135, Leu198, Thr199, Thr200, and Pro202. The substituted 3-carboxy-amino-phenyl functionality was at van der Waals distance from Phe131, Gly132, and Val135. The bulky tricyclic fluorescein moiety was located at the rim of the active site, on the protein surface, and strongly interacted with the alpha-helix formed by residues Asp130-Val135. All these interactions were preserved in the hCA IX-1 adduct, but the carbonyl moiety of the fluorescein tail of 1 participates in a strong hydrogen bond with the guanidine moiety of Arg130, an amino acid characteristic of the hCA IX active site. This may account for the roughly 2 times higher affinity of 1 for hCA IX over hCA II and may explain why in vivo the compound specifically accumulates only in hypoxic tumors overexpressing CA IX and not in the normal tissues. The compound is in clinical studies as an imaging tool for acute hypoxic tumors.