Entrance-channel plugging by natural sulfonamide antibiotics yields isoform-selective carbonic anhydrase IX inhibitors: an integrated in silico/ in vitro discovery of the lead SB-203207

Carbonic anhydrase IX (CA IX) is a hypoxia-induced pH regulator whose over-expression drives tumor progression and therapy resistance. Most CA inhibitors rely on zinc chelation and lack isoform selectivity, limiting clinical utility. Here we combined structure-based docking, 200 ns molecular-dynamics simulations and steady-state enzyme kinetics to assess four rare sulfonamide/sulfone natural products (altemicidin, SB-203207, SB-203208 and sulfadixiamycin A) as non-classical CA IX blockers. Docking located every ligand at the mouth of the catalytic funnel (- 7.2 to - 9.4 kcal mol⁻¹) without coordinating Zn²⁺. MD-derived free-energy landscapes and MM/PBSA calculations confirmed durable entrance-bound complexes for SB-203207/208 and sulfadixiamycin A (ΔG_total ≈ - 24 to - 27 kcal mol⁻¹) but frequent dissociation of altemicidin ( ≈ - 2 kcal mol⁻¹). Per-residue-decomposition pinpointed a hydrophobic wall (Leu91, Val121, Phe131, Leu198, Pro202, Phe243) plus anchoring H-bonds to Thr199 and Gln92. Recombinant-enzyme assays validated these predictions: SB-203207, SB-203208 and sulfadixiamycin A inhibited CA IX esterase activity with IC₅₀ = 73 ± 1, 99 ± 2, and 114 ± 3 nM, respectively, versus 41 ± 1 nM for reference acetazolamide. Crucially, SB-203207 showed marked selectivity, with SI = 28 (hCA I/IX) and SII = 14 (hCA II/IX), far exceeding the > 10-fold benchmark; SB-203208 and sulfadixiamycin A also met this threshold, whereas altemicidin was both weaker (1.90 µM) and less selective. Steady-state esterase kinetics were consistent with non-competitive inhibition and K_i values that mirrored the IC₅₀ rank order. SwissADME/ADMETlab profiling highlighted SB-203207 as the most developable hit. Together, these results establish entrance-channel plugging as an alternative mechanism for CA IX inhibition, identify SB-203207 as a potent and isoform-selective lead.