Sustainable leads for hypoxic tumor therapy: allosteric selective inhibition of carbonic anhydrase IX by abietane-type resin acids

Abstract

Carbonic anhydrase IX (CA IX) helps hypoxic tumors maintain the acidic extracellular milieu that drives invasion and therapy resistance, making it an attractive but still under-exploited drug target. Here we report that four abietane-type resin acids—callitrisic, levopimaric, palustric and pimaric acids—act as potent, non-competitive and CA IX-selective inhibitors. Virtual screening of 2400 natural products, followed by AutoDock Vina, positioned the rigid diterpenoid cores in a hydrophobic cleft that flanks—but does not overlap—the catalytic zinc. Stopped-flow CO₂-hydrase assays confirmed sub-micromolar potency, with IC₅₀ values of 93.4 ± 1.7 nM (callitrisic), 147.6 ± 4.7 nM (levopimaric), 290.5 ± 15.7 nM (palustric) and 340.7 ± 6.4 nM (pimaric); acetazolamide gave 44 ± 1.7 nM under identical conditions. Lineweaver–Burk and Michaelis–Menten plots revealed an allosteric, non-competitive mechanism, while counterscreens against housekeeping hCA I and hCA II yielded selectivity indices of 5–15, underscoring a therapeutic bias toward the tumor isoform. Two-hundred-nanosecond molecular-dynamics simulations produced single-basin free-energy landscapes dominated by van-der-Waals contacts with Phe243, Ala245, Pro248 and Ala249; MM/PBSA ranked binding free energies in the same order as enzymatic potency, with callitrisic acid reaching −20.58 ± 1.86 kcal mol⁻¹. Global MD metrics (RMSD, Rg, SASA, RMSF) demonstrated that ligand binding leaves the CA IX scaffold intact. In silico ADMET profiling predicted high oral and blood–brain permeability, low hERG liability and manageable CYP2C9/2C19 inhibition, highlighting log P moderation as the main optimization vector. Together, these findings establish abietane resin acids—particularly callitrisic acid—as sustainable, tractable leads for selectively disabling CA IX and normalizing tumor pH, paving the way for further optimization and in vivo validation.