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

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.