Selective inhibition of CA IX over CA XII using benzene sulfonamides: Disconnect between CA activity and growth inhibition in breast cancer cells

Carbonic anhydrases (CAs) have been implicated in tumor progression particularly membrane bound CA isoform IX (CA IX). In contrast to CA IX, expression of CA XII, specifically in breast cancer, is associated with a better outcome despite performing the same catalytic function. In this study, we have structurally modeled the orientation of bound ureido-substituted benzene sulfonamides (USBs) within the active site of CA XII, in comparison to CA IX and cytosolic off-target CA II, to understand isoform specific inhibition. This has identified specific residues within the CA active site, which differ between isoforms that are important for inhibitor binding and isoform specificity. The affinity of these inhibitors are two to three orders of magnitude lower in breast cancer cells, that selectively express CA IX or CA XII, relative to recombinant CA proteins. However, we show significantly greater inhibition of CA IX activity over CA XII. While the inhibitors block cell growth, without inducing cell death, this occurs at two orders of magnitude above the Ki values for inhibition of CA IX and CA XII in their respective cell types. Surprisingly, the USBs inhibited cell growth even in cells in which CA IX and CA XII expression was ablated. Despite the potential for these sulfonamides as chemotherapeutic agents, these data suggest that we reconsider the role of CA activity on growth inhibition.