Structural and Pharmacological Characterization of AT-121 Reveals Carbonic Anhydrase Inhibition as a Complementary Mechanism to Dual MOR/NOR Agonism.

AT-121 is a bifunctional ligand acting as a partial agonist at both μ-opioid and nociceptin receptors, designed to retain potent analgesia while reducing opioid-associated side effects. Carbonic anhydrases (CAs) catalyze the reversible conversion of carbon dioxide to bicarbonate and protons and, in the central nervous system, play key roles in neuronal excitability, synaptic transmission, and pain processing. Inhibition profiling across all human CA isoforms demonstrated activity against several cytosolic enzymes, as well as membrane-associated isoforms hCA IV and XII, the latter showing the highest inhibitory potency (KI = 70.1 nM). X-ray crystallography of the hCA II-AT-121 complex highlighting dual binding conformations mediated by an aliphatic sulfamide moiety within the enzyme active site. Complementary in silico docking simulations across additional isoforms supported these observations. Collectively, these findings suggest that AT-121 operates a multimodal mechanism combining dual opioid receptor activation with CA inhibition, supporting its potential as a safer, nonaddictive analgesic strategy.