Design of a Carbonic Anhydrase IX Active-Site Mimic To Screen Inhibitors for Possible Anticancer Properties

Abstract

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 18 O-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.Carbonic anhydrases (CAs) 1 are zinc-metalloenzymes that catalyze the reversible interconversion of CO 2 and HCO 3 -(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 † This work was supported by a grant (GM25154 to D.N.S. and R.M.) from the National Institutes of Health and the Maren Foundation (to R.M.). ‡ Coordinates and structure factors have been deposited in the Protein Data Bank as 3DC9.pdb and 3DC9.sf, 3DCS.pdb and 3DCS.sf, 3DCC.pdb and 3DCC.sf, 3DC3.pdb and 3DC3.sf, 3DCW.pdb and 3DCW.sf, 3DBU.pdb and 3DBU.sf, 3DAZ.pdb and 3DAZ.sf, 3D9Z.pdb and 3D9Z.sf, 3DD0.pdb and 3DD0.sf, and 3D8W.pdb and 3D8W.sf.

Saccharin: A lead compound for structure-based drug design of carbonic anhydrase IX inhibitors

“…The CA IX-mimic used for this study was designed based on previous studies by Genis, et al 20 (containing two active site amino acid substitutions) and reconstructed by Pinard et al 21 The CA IX-mimic utilized the easily crystallizable CA II as structural scaffold, with 7 point mutations in the active site to create a CA IX catalytic domain mimic as a model for structural analysis of ligand binding. Active site mutations in the CA IX-mimic include; A65S, N67Q, E69T, I91L, F131V, K170E, and L204A.…”

“…To circumvent this issue and gain insights into the specific binding interactions of both SAC and a SAC-based compound to CA IX, we use a CA IX-mimic. The CA IX-mimic is a CA II template with selective amino acid replacements that reflect a CA IX active site, which is easily expressed and crystallized like CA II but structurally analogous to the catalytic domain of CA IX (∼80% sequence identity between active site residues) 20 . The CA II to CA IX-mimic substitutions are A65S, N67Q, E69T, I91L, F131V, K170E, and L204A 21 .…”

Novel estradiol analogue induces apoptosis and autophagy in esophageal carcinoma cells

“…Carbonic anhydrases are zinc enzymes that control the CO 2 /bicarbonate ratio in the blood, and thus control the acid/base balance in the body [10,[24][25][26]. CAIX is overexpressed in a variety of tumors and contributes to the acidification of the extracellular environment of the tumors due to the conversion of carbon dioxide and water to carbonic acid [24,27,28]. The acidic environment promotes the expression of proteinases contributing to tumor growth, survival and metastasis [24,27,28].…”

“…CAIX is overexpressed in a variety of tumors and contributes to the acidification of the extracellular environment of the tumors due to the conversion of carbon dioxide and water to carbonic acid [24,27,28]. The acidic environment promotes the expression of proteinases contributing to tumor growth, survival and metastasis [24,27,28]. A comparison of the chemical structures of ESE-16 and 2ME reveals an exchange of a sulfamoylated group for a hydroxyl group at position 3 and the removal of a hydroxyl group at position 17 on ESE-16 ( Fig.…”

Carbonic anhydrase inhibitors: Design, synthesis and structural characterization of new heteroaryl-N-carbonylbenzenesulfonamides targeting druggable human carbonic anhydrase isoforms

“…The overexpression of hCA IX induces the pH imbalance of tumor issue contributing significantly to the extracellular acidification of solid tumor; thereby hCA IX inhibitors could specifically bind hypoxic tumor cells expressing this isoform; consequently they have been proposed as antitumor agents. [12][13][14][15][16][17][18][19][20][21] Finally, hCA XIV is a transmembrane isozyme with extracellularly oriented active site; it is highly abundant in neurons and axons in the murine and human brain, where it seems to play an important role in modulating excitatory synaptic transmission. [22] The catalytic domain of all human CA contains a common motif including a Zinc (II) ion that is essential for catalysis and is located at the bottom of a deep cleft.…”