Design of a Carbonic Anhydrase IX Active-Site Mimic to Screen Inhibitors for Possible Anti-Cancer Properties

摘要

Recently a convincing body of evidence has accumulated, suggesting that the over-expression of carbonic anhydrase isozyme IX (CA IX) in some cancers contributes to the acidification of the extracellular matrix, which in turn promotes the growth and metastasis of the tumor. These observations have made CA IX an attractive drug target for the selective treatment of certain cancers. Currently there is no available x-ray crystal structure of CA IX and this has hampered the rational design of selective CA IX inhibitors. In light of these observations and based on structural alignment homology, using the crystal structure CA II and the sequence of CA IX, a double mutant of CA II with Ala 65 replaced by Ser and Asn 67 replace by Gln has been constructed to resemble the active site of CA IX. This CA IX mimic has been characterized kinetically using ¹⁸O-exchange and structurally using x-ray crystallography, alone and in complex with five CA sulfonamide based inhibitors; acetazolamide, benzolamide, chlorzolamide, ethoxzolamide, and methazolamide, and compared to CA II. This structural information has been evaluated in relationship to inhibition studies and in vitro cytotoxicity assays and shows a correlated structure-activity relationship. Kinetic and structural studies of CA II and CA IX mimic reveal chlorzolamide to be a more potent inhibitor of CA IX inducing an active site conformational change upon binding. Additionally, chlorzolamide appears to be cytotoxic to prostate cancer cells. This preliminary study demonstrates that the CA IX mimic may provide a useful model to design more isozyme specific CA IX inhibitors which may lead to development of new therapeutic treatments of some cancers.