Design of a carbonic anhydrase IX active-site mimic to screen inhibitors for possible anticancer properties

Carbonic anhydrases (CAs)1 are zinc-metalloenzymes that catalyze the reversible inter-conversion of CO2 and HCO3- (1). Since their discovery, the CAs have been extensively studied due to their important physiological functions in all kingdoms of life. This family of enzymes is broadly comprised of three well-studied, structurally distinct (α, β, and γ) classes. The α–class is present in vertebrates, but have also been shown to present in other organisms. They (and α-CA domains in more complex isoforms) have a molecular weight of ~29kDa. There are 14 expressed α-CAs (CA I - XIV) in humans, and the active CAs play roles in respiration, pH homeostasis, fluid production, and other functions as yet to be determined (2-5). The α-CAs all share the same overall mixed α/β fold with approximate dimensions of 50 × 40 × 40 A3. The active site is characterized by a conical cavity that is approximately 15 A deep. The zinc ion is located at the bottom of the conical active site cavity and is tetrahedrally coordinated by three histidine ligands and a bound hydroxide/water (1). The active sites between isoforms are nearly identical other than a few amino acids that line the cavity (6). CA IX is a unique member of the human α-class CAs, as it is a membrane associated glycoprotein, composed of several domains including a short intracellular region, a single transmembrane helix, and an extracellular proteoglycan domain that encodes a catalytic CA domain (7). Under normal conditions CA IX is commonly expressed in cells that are thought to need to maintain extracellular pH, such as gastric mucosal cells. However, in many cancers it is over-expressed as a result of hypoxia(3). The regulation of the CA IX gene has been shown to be controlled by the hypoxia inducing factor-1 (7). It has been hypothesized that as tumor growth progresses and becomes insufficient to maintain a supply of oxygen, the cancer cell remodels metabolically, which is partially achieved by the up-regulation of CA IX. Therefore CA IX is considered to be a marker of tumor hypoxia (8). In hypoxic tumors, it is believed that CA IX plays a critical role in cell survival. In tumors there in an observed increase in CO2 concentration (9, 10). It is believed that this is not a result of oxidative metabolism, but rather a by-product of an increase in the pentose phosphate pathway. This serves to replenish the supply of NADPH and generate ribose-5-phosphate, necessary for nucleotide and coenzyme production (11). The surplus of CO2 is converted to HCO3- and a proton by CA IX, creating the significant increase in extracellular proton concentration causing the acidification of the tumor microenvironment. The alteration in proton flux is also believed to affect the activity of ion transporters and channels (5). Additionally, the acidity may cause the exclusion of weakly basic chemotherapeutic agents rendering traditional therapies less effective (7). The proteoglycan domain of CA IX has been implicated in the disruption of cell-cell adhesion by breaking the connection of E-cadherin to the cytoskeleton, which may lead to tumor invasion (12). Several studies have shown that inhibition of CA IX can lead to decreased invasiveness as well as inducing cell death under hypoxic conditions (3, 8, 13-16). These factors, taken together, provide strong evidence to suggest that CA IX might be an attractive drug target for the treatment of cancers. One significant barrier that needs to be overcome, for the development of CA IX inhibitors as effective cancer treatments, is to produce a CA IX isoform specific inhibitor, which has a significantly higher affinity for CA IX than other active CA isoforms. The development of a high affinity CA IX inhibitor has been hampered due to the lack of an available crystal structure of CA IX. CA IX being a membrane protein and has so far proven itself difficult to express in sufficient soluble and properly folded quantities for crystallization (17, 18). To overcome these issues, this research reports the expression and structural and kinetics studies of a CA IX active site mimic. The design of the CA IX mimic based on a structural alignments comparison with CA II, and is a double mutant of CA II (A65S N67Q CA II) that imitates the active site of CA IX. This CA IX mimic was expressed and characterized both kinetically and crystallographically, alone and in complex with several common sulfonamide inhibitors, acetazolamide, benzolamide, chlorzolamide, ethoxzolamide, and methazolamide. Preliminary data reveals that the inhibition profiles of the CA IX mimic mirror wild type CA IX compared to values previously published (17). In addition, the crystal structures of the CA IX mimic bound to these drugs reflect the kinetics, showing chlorzolamide, the drug with greatest affinity to CA IX and the CA IX mimic undergoes active site conformational changes rather than a simple lock and key model of inhibition. Further this structural information has been evaluated in relationship to inhibition studies and in vitro cytotoxicity assays and shows a correlated structure-activity relationship. To our knowledge this is the first time a protein mimic has been engineered from a related isozyme for the purpose of drug design. The CA IX mimic presented provides a unique opportunity in developing and assessing CA IX isoform specific inhibitors, while on-going studies attempt to obtain the crystal structure of the catalytic domain of CA IX.