Targeting carbonic anhydrase IX activity and expression

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.

Figure 1

(A) Schematic diagram of CA IX structure. Proteoglycan-like domain (PG, yellow), catalytic domain (CA, violet), a transmembrane anchor (TM, cyan), and an intracellular domain (IC, green). Spheres represent glycosylation sites. The PG domain was generated using a structure prediction server Robetta [24] and the CA domain is from the coordinates of the CA IX crystal structure (PDB ID: 3IAI). The TM anchor and IC domain were generated using Chimera [25] and COOT [26] software packages, respectively. This figure was adapted from: Mahon et al. [27] (B) Acetazolamide (AZM) bound in the active site of CA IX (PDB ID: 3IAI). Figure was created using PyMol [28].

Figure 2

CA IX expression in adult human tissue. Note that (*) indicates high-grade tumor tissues.

Figure 2

CA IX expression in adult human tissue. Note that (*) indicates high-grade tumor tissues.

Figure 3

Surface rendition of CA IX (PDB ID: 3IAI). Residues in the hydrophobic (red) and hydrophilic (blue) cleft are as labeled; residues conserved (white) and those that differ (yellow) between CA II and CA IX. Figure made using PyMol [28].

Figure 4

The active site of CA II (PDB ID: 3KS3). The zinc ion is represented by a grey sphere and is coordinated by His 94, 96 and 119, and a H₂O/OH⁻ molecule. The zinc bound H₂O/OH⁻ binds at a distance of 1.9 Å away from the zinc ion. Water (W) are represented by red spheres while hydrogen bonds (H-bonds, Å) are represented by dotted lines. This figure was made using PyMol [28].

Figure 5

CAIs designed to target the extracellular CA domain of CA IX as compared to classic inhibitors. (A) Classic (B) Bulky (C) Fluorescent (D) Cationic sulfonamides (E) Glycoconjugates all show extensive differences in terms of CA IX specific targeting potential.(Figure was adapted from [67] and made using PyMol [28] and ChemDraw [68] software packages).

Figure 6

Prodrug CAIs. (A) Passive prodrug CAIs (4-(2-mercaptophenylcarboxamido) benzenesulfonamide) utilizes the microenvironmental changes to become unmasked. (B) Active prodrug CAIs require the presence of esterase activity either from ubiquitous esterases or the weak esterase activity of CAs to become unmasked.

Figure 7

Pathway of AAV-mediated siRNA delivery to target CA expression in a tumor cell. The siRNA construct is delivered via a transduced AAV vector and its episome is trafficked to the nucleus. Within the nuclease the episomal DNA is transcribed by RNA pol II to produce a pre-shRNA construct that is further processed and transported to the cytosol where it becomes an siRNA duplex. The siRNA duplex is then processed by RISC and escorted to the specific mRNA region encoding for CA IX. Once bound, via complementary base-pairing, the CA IX mRNA is degraded and expression of CA IX is abolished. (This figure was adapted from [116] and AAV3B; PDB ID: 3KIC [117]).