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.

The alpha carbonic anhydrases (α-CAs) are a group of structurally related zinc metalloenzymes that catalyze the reversible hydration of CO2 to HCO3 −. 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.

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Serpins (serine protease inhibitors) are ubiquitous, complex, and highly active regulatory molecules that effectively control multiple coagulation, inflammatory, and neuroendocrine pathways (1-3). The amino acid sequence in the reactive center loop (RCL) 3 of serpins acts as bait for target serine proteases, initiating structural changes in the serpin-protease complex and culminating in suicide inhibition (1-3). This same RCL can insert into the neighboring ␤-sheet A in other serpins in serpinopathies, causing serpin aggregates induced by genetic mutations and causing disease as for anti-thrombin III (SERPIN C1, ATIII), ␣-1 antitrypsin (SERPIN A1, AAT), and neuroserpin (SERPIN I1, NSP). Whereas the amino acid residues in the RCL provide target P1-P1Ј sequences, referred to as a scissile bond, serpins also require the greater part of the protein structure to function with true serpin-protease inhibitory activity (4, 5). However, as for other proteins, peptides derived during protein metabolism may act to extend serpin activity beyond the initial suicide-inhibitory function, both increasing and decreasing responses (6 -9). In prior work, significant and prolonged antiinflammatory functions have been detected with myxomavirus-derived Serp-1 (10 -17) and mammalian serpin NSP purified protein injections in animal models of vascular disease (18,19). We have hypothesized that peptides produced by protease cleavage of the RCL sequence during natural proteolytic metabolism of Serp-1 or NSP may extend serpin activity, increasing anti-inflammatory activity after serpin-protease complex formation. Thus, these serpin RCL peptide metabolites have the theoretical potential to interfere with either protease activity by acting as a protease bait or inhibitors or to inhibit other serpins by inserting into the ␤-sheet.Many proteins have active metabolites providing additional and/or expanded functions. Peptides derived from calreticulin (20) and apolipoprotein E (Ep1B) (21) have proven anti-atherogenic activity, reducing inflammation and plaque growth in animal models. Serpins also have reported active terminal peptide metabolites. Among the serpins, angiotensinogen is a protein with serpin structure but lacking serpin inhibitory activity (6).