Discovery and Characterization of Novel Selective Inhibitors of Carbonic Anhydrase IX

Abstract

Human carbonic anhydrase IX (CA IX) is highly expressed in tumor tissues, and its selective inhibition provides a potential target for the treatment of numerous cancers. Development of potent, highly selective inhibitors against this target remains an unmet need in anticancer therapeutics. A series of fluorinated benzenesulfonamides with substituents on the benzene ring was designed and synthesized. Several of these exhibited a highly potent and selective inhibition profile against CA IX. Three fluorine atoms significantly increased the affinity by withdrawing electrons and lowering the pKa of the benzenesulfonamide group. The bulky ortho substituents, such as cyclooctyl or even cyclododecyl groups, fit into the hydrophobic pocket in the active site of CA IX but not CA II, as shown by the compound's co-crystal structure with chimeric CA IX. The strongest inhibitor of recombinant human CA IX's catalytic domain in human cells achieved an affinity of 50 pM. However, the high affinity diminished the selectivity. The most selective compound for CA IX exhibited 10 nM affinity. The compound that showed the best balance between affinity and selectivity bound with 1 nM affinity. The inhibitors described in this work provide the basis for novel anticancer therapeutics targeting CA IX.

Intrinsic Thermodynamics and Structures of 2,4‐ and 3,4‐Substituted Fluorinated Benzenesulfonamides Binding to Carbonic Anhydrases

“…We also showed that the extremely high binding affinity of tetrafluorobenzenesulfonamide derivatives to CA I was associated with strong enthalpic interactions. We have modified 4‐substituted 2,3,5,6‐tetrafluorobenzenesulfonamides by introducing different substituents at the ortho or meta positions and found that substitutions with highly hydrophobic groups at these positions led to more potent and selective CA IX inhibitors by lowering the affinity toward the off‐target isoforms CA I and CA II . Fluorine atoms on the first benzene ring increase the observed binding affinity by withdrawing electrons and lowering the p K a value of the sulfonamide group, whereas the bulky hydrophobic groups of the compounds at the ortho or meta positions made favorable hydrophobic contacts with the amino acids of CA IX.…”

“…The crystal structures of CA I– 10 h , CA XIII– 10 h , and CA II– 2 m contained two subunits in the asymmetric unit, whereas the structures of CA II– 1 b , CA II– 10 n , and CA II– 10 d contained one protein chain in the asymmetric unit. The benzene ring of the fluorinated benzenesulfonamides in the crystal structures with CA II and CA XII can be found in two positions, as previously described . The benzene ring can be located in the same plane as the nitrogen atom of the sulfonamide group, and such orientation will be referred to as the “in‐plane” orientation (Figure S3 A in the Supporting Information).…”

“…However, CA IX has been shown in many studies to be the most inducible protein under the hypoxic conditions present in solid tumors, where CA IX functions in pH regulation and participates in extracellular acidification by proton production; it thus promotes tumor aggressiveness . To date, many low‐molecular‐weight compounds have been studied that bind to CA IX and CA XII and inhibit the enzymatic activity . In vivo studies have shown that the inhibition of CA IX is effective in tumor growth suppression.…”

Prognostic relevance of carbonic anhydrase IX expression is distinct in various subtypes of breast cancer and its silencing suppresses self-renewal capacity of breast cancer cells

“…Interestingly, CAIX was found to contribute to cell migration and invasion by facilitating formation of focal adhesions, ion transport and pH control at the leading edge of lamellipodia of moving cells [27,28]. Currently, substantial effort is devoted to the development of potent, cancer-selective CAIX inhibitors, including monoclonal antibodies [29] and small molecule inhibitors [30][31][32].…”

Proteochemometric Modeling of the Interaction Space of Carbonic Anhydrase and its Inhibitors: An Assessment of Structure‐based and Sequence‐based Descriptors

“…Previous studies have confirmed the critical role of F131 in hCA II. It has been shown that the presence of an aromatic residue with a bulky side chain instead of an aliphatic one can cause different patterns of binding ,,. In our analyses, we noticed that the spatial distance between side chains of V121 and F131 in hCA II is highly compatible with that of DRY‐DRY descriptor (∼7.5 Å).…”