Crystal structure of the catalytic domain of the tumor-associated human carbonic anhydrase IX

Abstract

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.

Deciphering the Mechanism of Carbonic Anhydrase Inhibition with Coumarins and Thiocoumarins

“…However, a large number of derivatives showed effective or very effective inhibitory activity against this isoform, which represents a new drug target for developing antitumor therapies or diagnostic agents. 7,9 Thus, thiocoumarin 17 and several coumarins (18,19,21, and 23) showed low nanomolar affinity for this enzyme, with inhibition constants in the range of 45-98 nM. These coumarins incorporate the 6-hydroxymethyl-and 3-ester moieties (18 and 19), with no important differences of activity between the methyl and ethyl esters in this case.…”

Ureido-Substituted Benzenesulfonamides Potently Inhibit Carbonic Anhydrase IX and Show Antimetastatic Activity in a Model of Breast Cancer Metastasis

“…[1][2][3] In this latter case, only the transmembrane, tumor-associated isozymes (IX and XII) should be inhibited, as CA II may have the function of housekeeping enzyme and its inhibition may lead to side effects. [1][2][3] The following structure-activity relationship (SAR) can be observed from data of Table 1 for the series of ureidosulfonamides 4-30 investigated here.…”

“…[1][2][3][4][5][6] CA IX is the most strongly overexpressed gene in response to hypoxia in human cancer cells. [2][3][4] This enzyme is a multidomain protein 2 with the CA subdomain situated outside the cell and possessing a very high CO 2 hydrase catalytic activity, 7 making it a key player in the regulation of tumor pH.…”

“…[1][2][3][4][5][6] CA IX is the most strongly overexpressed gene in response to hypoxia in human cancer cells. [2][3][4] This enzyme is a multidomain protein 2 with the CA subdomain situated outside the cell and possessing a very high CO 2 hydrase catalytic activity, 7 making it a key player in the regulation of tumor pH. 1-6 CA IX expression is strongly increased in many types of solid tumors, such as gliomas/ependymomas, mesotheliomas, and papillary/follicular carcinomas; carcinomas of the bladder, uterine cervix, kidneys, esophagus, lungs, head and neck, breast, brain, and vulva; and squamous/basal cell carcinomas.…”

Interfering with pH regulation in tumours as a therapeutic strategy

“…Interestingly, CA9 is the most strongly overexpressed gene in response to hypoxia in human cancer cells 28,30 , and it is also the most active isoform of carbonic anhydrase for the car bon dioxide hydration reaction 29,31 . The X-ray crystal structure of CA9 reveals that it is a dimeric enzyme 32 , unlike all other carbonic anhydrase isoforms known so far, which are monomeric. Like CA9, CA12 is a transmembrane isoform with an extracellular active site 27 but it has lower catalytic activity than CA9 (REFS 15,33).…”