Carbonic anhydrase (CA) IX is a plasma membrane-associated member of the α-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 α-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 three-dimensional 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 α-CAs, further prospects for the rational drug design of isozyme-specific CA inhibitors are proposed, given that inhibition of this enzyme shows antitumor activity both in vitro and in vivo.

Carbonic anhydrase IX (CA IX) is an exceptional member of the CA protein family; in addition to its classical role in pH regulation, it has also been proposed to participate in cell proliferation, cell adhesion, and tumorigenic processes. To characterize the biochemical properties of this membrane protein, two soluble recombinant forms were produced using the baculovirus-insect cell expression system. The recombinant proteins consisted of either the CA IX catalytic domain only (CA form) or the extracellular domain, which included both the proteoglycan and catalytic domains (PG + CA form). The produced proteins lacked the small transmembrane and intracytoplasmic regions of CA IX. Stopped-flow spectrophotometry experiments on both proteins demonstrated that in the excess of certain metal ions the PG + CA form exhibited the highest catalytic activity ever measured for any CA isozyme. Investigations on the oligomerization and stability of the enzymes revealed that both recombinant proteins form dimers that are stabilized by intermolecular disulfide bond(s). Mass spectrometry experiments showed that CA IX contains an intramolecular disulfide bridge (Cys¹¹⁹-Cys²⁹⁹) and a unique N-linked glycosylation site (Asn³⁰⁹) that bears high mannose-type glycan structures. Parallel experiments on a recombinant protein obtained by a mammalian cell expression system demonstrated the occurrence of an additional O-linked glycosylation site (Thr⁷⁸) and characterized the nature of the oligosaccharide structures. This study provides novel information on the biochemical properties of CA IX and may help characterize the various cellular and pathophysiological processes in which this unique enzyme is involved.

Carbonic anhydrases (CAs, EC 4.2.1.1) exist as five genetically distinct families (α, β, γ, δ and ζ) in organisms all over the phylogenetic tree. Due to the ubiquity of such enzymes, the selective inhibition and polypharmacology of inhibitors is an important aspect of all drug design campaigns. There are several classes of CA inhibitors (CAIs): i) metal ion binders (sulfonamides and their isosteres [sulfamates/sulfamides], dithiocarbamates, mercaptans and hydroxamates); ii) compounds anchoring to the zinc-coordinated water molecule/hydroxide ion (phenols, carboxylates, polyamines, esters and sulfocoumarins) and iii) coumarins and related compounds which apparently bind even further away from the metal ion. The authors rationalize the drug design strategies of inhibitors belonging to the first two classes, based on recent X-ray crystallographic data. More precisely, this is achieved by analyzing how the hydrophobic and hydrophilic halves of the enzyme active site interact with inhibitors. This task has been eased by the recent report of β-CA-like enzymes possessing carbon disulfide and carbonyl sulfide hydrolase activities, respectively, allowing the authors to propose a general approach of structure-based drug design of CAIs. 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 anhydrases (EC 4.2.1.1) IX (hCA IX) and XII (hCA XII) are two tumor-associated proteins, being overexpressed in many tumors and involved in critical processes associated with cancer progression and response to therapy. Both are multi-domain proteins consisting of an extracellular catalytic domain (CA), a transmembrane portion (TM) and an intracytoplasmic (IC) segment. These domains have peculiar biochemical and physiological features. CA IX contains an additional proteoglycan-like (PG) domain at the N-terminus which constitutes a unique feature of this enzyme within the CA family. Starting from a brief description of the main molecular and catalytic features of both enzymes, their role in tumor physiology and their three-dimensional structure, this review describes the main classes of small molecule inhibitors, investigated between 2008 and 2013, able to inhibit these enzymes for both diagnostic and therapeutic applications. A consistent number of patents on molecules able to inhibit the catalytic activity of CA IX and CA XII have been recently reported. Most patents deal with classical sulfonamide derivatives, demonstrating that introducing suitable substituents on the inhibitor scaffold, good selectivity can be obtained. However, the most impressive results are related to compounds containing novel chemotypes, such as coumarins and thiocumarins. Thus, it is expected that research in next future will be more dedicated to the development of molecules containing new chemotypes and a large number of studies in such field have already been published demonstrating the role of these enzymes in carcinogenesis and metastases formation.