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.

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The anticancer activities and SARs of estradiol-17-O-sulfamates and estradiol 3,17-O,O-bis-sulfamates (E2bisMATEs) as steroid sulfatase (STS) inhibitors and antiproliferative agents are discussed. Estradiol 3,17-O,O-bis-sulfamates 20 and 21, in contrast to the 17-O-monosulfamate 11, proved to be excellent STS inhibitors. 2-Substituted E2bisMATEs 21 and 23 additionally exhibited potent antiproliferative activity with mean graph midpoint values of 18-87 nM in the NCI 60-cell-line panel. 21 Exhibited antiangiogenic in vitro and in vivo activity in an early-stage Lewis lung model, and 23 dosed p.o. caused marked growth inhibition in a nude mouse xenograft tumor model. Modeling studies suggest that the E2bisMATEs and 2-MeOE2 share a common mode of binding to tubulin, though COMPARE analysis of activity profiles was negative. 21 was cocrystallized with carbonic anhydrase II, and X-ray crystallography revealed unexpected coordination of the 17-O-sulfamate of 21 to the active site zinc and a probable additional lower affinity binding site. 2-Substituted E2bisMATEs are attractive candidates for further development as multitargeted anticancer agents.