Carbonic anhydrase IX: Biochemical and crystallographic characterization of a novel antitumor target

ArticleinBiochimica et Biophysica Acta 1804(2):404-9 · September 2009with8 Reads
DOI: 10.1016/j.bbapap.2009.07.027 · Source: PubMed
Abstract
Isoform IX of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), CA IX, is a transmembrane protein involved in solid tumor acidification through the HIF-1alpha activation cascade. CA IX has a very high catalytic activity for the hydration of carbon dioxide to bicarbonate and protons, even at acidic pH values (of around 6.5), typical of solid, hypoxic tumors, which are largely unresponsive to classical chemo- and radiotherapy. Thus, CA IX is used as a marker of tumor hypoxia and as a prognostic factor for many human cancers. CA IX is involved in tumorigenesis through many pathways, such as pH regulation and cell adhesion control. The X-ray structure of the catalytic domain of CA IX has been recently reported, being shown that CA IX has a typical alpha-CA fold. However, the CA IX structure differs significantly 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 proteoglycan (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. As all mammalian CAs, CA IX is inhibited by several main classes of inhibitors, such as the inorganic anions, the sulfonamides and their bioisosteres (sulfamates, sulfamides, etc.), the phenols, and the coumarins. The mechanism of inhibition with all these classes of compounds is understood at the molecular level, but the sulfonamides and their congeners have important applications. It has been recently shown that both in vitro, in cell cultures, as well as in animals with transplanted tumors, CA IX inhibition with sulfonamides lead to a return of the extracellular pH to more normal values, which leads to a delay in tumor growth. As a consequence, CA IX represents a promising antitumor target for the development of anticancer agents with an alternative mechanism of action.
    • "CA IX consists of a large extracellular part, containing a unique N-terminal proteoglycan-like region (PG), and a carbonic anhydrase domain which accelerates CO 2 –HCO 3 interconversion 105–106-fold over the uncatalyzed reactions. This extracellular domain is anchored to the cell membrane by a single pass transmembrane region connected to a short intracellular (IC) domain [36]. The PG domain appears to play some role in cell adhesion characteristics of malignant cells leading them to more easily separate from the primary tumor and travel to attach elsewhere and begin metastatic growth [169]. "
    [Show abstract] [Hide abstract] ABSTRACT: Hypoxia, depending upon its magnitude and circumstances, evokes a spectrum of mild to severe acid–base changes ranging from alkalosis to acidosis, which can alter many responses to hypoxia at both non-genomic and genomic levels, in part via altered hypoxia-inducible factor (HIF) metabolism. Healthy people at high altitude and persons hyperventilating to non-hypoxic stimuli can become alkalotic and alkalemic with arterial pH acutely rising as high as 7.7. Hypoxia-mediated respiratory alkalosis reduces sympathetic tone, blunts hypoxic pulmonary vasoconstriction and hypoxic cerebral vasodilation, and increases hemoglobin oxygen affinity. These effects and others can be salutary or counterproductive to tissue oxygen delivery and utilization, based upon magnitude of each effect and summation. With severe hypoxia either in the setting of profound arterial hemoglobin desaturation and reduced O2 content or poor perfusion (ischemia) at the global or local level, metabolic and hypercapnic acidosis develop along with considerable lactate formation and pH falling to below 6.8. Although conventionally considered to be injurious and deleterious to cell function and survival, both acidoses may be cytoprotective by various anti-inflammatory, antioxidant, and anti-apoptotic mechanisms which limit total hypoxic or ischemic–reperfusion injury. Attempts to correct acidosis by giving bicarbonate or other alkaline agents under these circumstances ahead of or concurrent with reoxygenation efforts may be ill advised. Better understanding of this so-called “pH paradox” or permissive acidosis may offer therapeutic possibilities. Rapidly growing cancers often outstrip their vascular supply compromising both oxygen and nutrient delivery and metabolic waste disposal, thus limiting their growth and metastatic potential. However, their excessive glycolysis and lactate formation may not necessarily represent oxygen insufficiency, but rather the Warburg effect—an attempt to provide a large amount of small carbon intermediates to supply the many synthetic pathways of proliferative cell growth. In either case, there is expression and upregulation of many genes involved in acid–base homeostasis, in part by HIF-1 signaling. These include a unique isoform of carbonic anhydrase (CA-IX) and numerous membrane acid–base transporters engaged to maintain an optimal intracellular and extracellular pH for maximal growth. Inhibition of these proteins or gene suppression may have important therapeutic application in cancer chemotherapy.
    Full-text · Chapter · Jan 2016 · Molecules
    • "Not only do these compounds exhibit high affinity for CA IX/XII but the bulky sugar moieties cause a reduction in membrane permeability allowing for selective targeting of the extracellular facing catalytic domain of both tumor associated isoforms thus acting as location specific CAIs119120121. Similar to adding bulky-carbohydrate moieties to sulfonamides , steroid sulfatase inhibitors, which have been designed based on previously seen antimitotic inhibitors [136, 137] are able to take advantage of the variable residues in the hydrophobic pocket of specific CAs via van Der Waals contacts of the steroidal backbone136137138. The same trend was seen in energy calculations from molecular docking studies of such compounds with CA IX [137]. "
    [Show abstract] [Hide abstract] ABSTRACT: The alpha carbonic anhydrases (α-CAs) are a group of structurally related zinc metalloenzymes that catalyze the reversible hydration of CO2 to HCO 3 - . Humans have 15 different α-CAs with numerous physiological roles and expression patterns. Of these, 12 are catalytically active, and abnormal expression and activities are linked with various diseases, including glaucoma and cancer. Hence there is a need for CA isoform specific inhibitors to avoid off-target CA inhibition, but due to the high amino acid conservation of the active site and surrounding regions between each enzyme, this has proven difficult. However, residues towards the exit of the active site are variable and can be exploited to design isoform selective inhibitors. Here we discuss and characterize this region of “selective drug targetability” and how these observations can be utilized to develop isoform selective CA inhibitors.
    Full-text · Article · Mar 2015
    • "In CA IX three histidine residues (His 226, 228 and 251, as numbered in the full length aa sequence) coordinate the zinc ion at the base of the active site cleft; in the crystal structure (PDB ID: 3IAI) acetazolamide (AZM) displaces a zinc bound water/hydroxide (Zn-OH/H2O) molecule maintaining a tetrahedral coordination about the zinc ion (Figure 1B). Variability between the CA isoforms occurs in the hydrophobic and hydrophilic pockets of the active site and surface amino acids [19,20,22] (Figure 2). The structural and amino acid conservation that exists between the active sites of human CA isoforms has made it difficult to design CA IX specific inhibitors and avoid off-target inhibition of other CAs that are ubiquitously expressed in normal tissue [23]. "
    [Show abstract] [Hide abstract] ABSTRACT: Metastatic tumors are often hypoxic exhibiting a decrease in extracellular pH (~6.5) due to a metabolic transition described by the Warburg Effect. This shift in tumor cell metabolism alters the tumor milieu inducing tumor cell proliferation, angiogenesis, cell motility, invasiveness, and often resistance to common anti-cancer treatments; hence hindering treatment of aggressive cancers. As a result, tumors exhibiting this phenotype are directly associated with poor prognosis and decreased survival rates in cancer patients. A key component to this tumor microenvironment is carbonic anhydrase IX (CA IX). Knockdown of CA IX expression or inhibition of its activity has been shown to reduce primary tumor growth, tumor proliferation, and also decrease tumor resistance to conventional anti-cancer therapies. As such several approaches have been taken to target CA IX in tumors via small-molecule, anti-body, and RNAi delivery systems. Here we will review recent developments that have exploited these approaches and provide our thoughts for future directions of CA IX targeting for the treatment of cancer.
    Full-text · Article · Feb 2015
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