Chemical Determinants of Spinal Analgesia in NTS2-Selective Macrocycles

Neurotensin (NT)-based ligands represent promising non-opioid analgesics. Here, we designed and synthesized a focused library of macrocyclic NT analogs combining substitutions previously shown to enhance NTS2 affinity, selectivity, and/or antinociceptive activity. Several macrocycles retained low-nanomolar NTS2 affinity and remarkable subtype selectivity, including analog M19, which exhibited >53,000-fold selectivity over NTS1, the highest reported to date. Although combining favorable substitutions did not produce additive improvements, M19 and M25 displayed potent spinal antinociceptive activity in rat tail-flick and formalin tests. Most importantly, removal of the N-terminal amine abolished antinociceptive activity while preserving high NTS2 affinity, identifying this pharmacophore as a critical determinant of NTS2-mediated analgesia. Molecular dynamics simulations comparing M25 with its desamino analog M29 revealed distinct extracellular interaction networks and conformational dynamics, including the loss of prominent ECL2-N-terminal and ECL3 interactions in M29. These findings provide a structural framework for the rational development of selective, non-opioid analgesics targeting NTS2.