Structure-activity relationships of contulakin-G: Design, synthesis, and characterization of Thr¹⁰ analogs for pain...

Contulakins are a subclass of conopeptides derived from cone snail venom, known for their analgesic properties mediated through activation of neurotensin receptors NTS1 and NTS2. Among them, contulakin-G, isolated from Conus geographus, exhibits a favorable pharmacokinetic profile, including a 24-hour plasma half-life, and produces potent opioid-independent antinociceptive effects. To facilitate synthesis while preserving analgesic efficacy, we investigated the structure-activity relationships of contulakin-G, focusing on modifications to the glycosylated Thr¹⁰ residue. In the first series, Thr¹⁰ was substituted by natural and non-natural amino acids encompassing diverse physiochemical and steric properties. Most substitutions had minimal impact on receptor affinity, though some increased the NTS2/NTS1 selectivity ratio. Of these, only the Tyr¹⁰ analog retained antinociceptive efficacy comparable to native contulakin-G. Building on tyrosine's hydrophobicity, a second series introduced non-natural hydrophobic residues at position 10, including Gln-cycloheptyl, Gln-biphenylmethyl, and Gln-dimethyladamantyl, along with a non-glycosylated form. In both tonic and postoperative pain models, the non-glycosylated and Gln-cycloheptyl analogs demonstrated potent analgesic activity, comparable to contulakin-G and reversed mechanical allodynia in a neuropathic pain model. In contrast, a third series involving N-terminal truncations led to reduced plasma half-life and loss of antinociceptive efficacy, underscoring the importance of the N-terminal region. Molecular modeling further revealed that full-length analogs engage a broader NTS2 binding interface than truncated derivatives. These findings support the development of synthetically accessible contulakin-G analogs as promising non-opioid analgesics.