Steroidal A/B-ring fusion as a strategy for isoform-selective inhibition of human carbonic anhydrases

Carbonic anhydrases (CAs) are zinc-containing metalloenzymes that catalyse the reversible hydration of carbon dioxide and thus play a crucial role in pH regulation. Among the isoforms of carbonic anhydrases, CA IX is a cancer-associated enzyme overexpressed in hypoxic tumours, which makes it an attractive target for anti-cancer drug development. Steroidal compounds, with their rigid frameworks and diverse functionalization potential, have emerged as promising scaffolds for designing selective CA inhibitors. Their ability to engage in specific enzyme interactions makes them valuable for developing selective inhibitors targeting medically relevant CA isoforms. This study explores the inhibitory activities and binding modes of four steroid skeletons-5β-steroid, estra-1,3,5(10)-triene, Δ5-steroid and 5α-steroid-in interaction with selected CA isoforms. Structural and inhibition studies have revealed that steroidal sulfamate derivatives effectively coordinate with the active site zinc ion, adopting distinct binding modes based on isoform-specific variations. The hydrophobic patch at the active site entrance, influenced by a key difference in the residue present at position 131 (Phe131 in CA II vs. Val131 in CA IX), plays a crucial role in modulating binding interactions. Estra-1,3,5(10)-triene derivatives exhibit nanomolar inhibition of both CA II and CA IX, demonstrating adaptability through alternative binding conformations. By contrast, Δ5-steroid compounds show enhanced selectivity towards CA IX and appear to be less easily accommodated by the more constrained active site of CA II. These findings highlight the potential of steroidal compounds as inhibitors of specific CA isoforms. In particular, estra-1,3,5(10)-triene and Δ5-steroid compounds without a C-17 substitution emerge as strong candidates for further development, targeting the cancer-associated CA IX and other medically relevant isoforms.