Helix-constrained nociceptin peptides are potent agonists and antagonists of ORL-1 and nociception

Rink-Jan Lohman, Rosemary S Harrison, Gloria Ruiz-Gómez, Huy N Hoang, Nicholas E Shepherd, Shiao Chow, Timothy A Hill, Praveen K Madala, David P Fairlie

Research output: Contribution to journalReview articlepeer-review

Abstract / Description of output

Nociceptin (orphanin FQ) is a 17-residue neuropeptide hormone with roles in both nociception and analgesia. It is an opioid-like peptide that binds to and activates the G-protein-coupled receptor opioid receptor-like-1 (ORL-1, NOP, orphanin FQ receptor, kappa-type 3 opioid receptor) on central and peripheral nervous tissue, without activating classic delta-, kappa-, or mu-opioid receptors or being inhibited by the classic opioid antagonist naloxone. The three-dimensional structure of ORL-1 was recently published, and the activation mechanism is believed to involve capture by ORL-1 of the high-affinity binding, prohelical C-terminus. This likely anchors the receptor-activating N-terminus of nociception nearby for insertion in the membrane-spanning helices of ORL-1. In search of higher agonist potency, two lysine and two aspartate residues were strategically incorporated into the receptor-binding C-terminus of the nociceptin sequence and two Lys(i)→Asp(i+4) side chain-side chain condensations were used to generate lactam cross-links that constrained nociceptin into a highly stable α-helix in water. A cell-based assay was developed using natively expressed ORL-1 receptors on mouse neuroblastoma cells to measure phosphorylated ERK as a reporter of agonist-induced receptor activation and intracellular signaling. Agonist activity was increased up to 20-fold over native nociceptin using a combination of this helix-inducing strategy and other amino acid modifications. An NMR-derived three-dimensional solution structure is described for a potent ORL-1 agonist derived from nociceptin, along with structure-activity relationships leading to the most potent known α-helical ORL-1 agonist (EC₅₀ 40 pM, pERK, Neuro-2a cells) and antagonist (IC₅₀ 7 nM, pERK, Neuro-2a cells). These α-helix-constrained mimetics of nociceptin(1-17) had enhanced serum stability relative to unconstrained peptide analogues and nociceptin itself, were not cytotoxic, and displayed potent thermal analgesic and antianalgesic properties in rats (ED₅₀ 70 pmol, IC₅₀ 10 nmol, s.c.), suggesting promising uses in vivo for the treatment of pain and other ORL-1-mediated responses.

Original languageEnglish
Pages (from-to)1-55
Number of pages55
JournalVitamins and Hormones
Publication statusPublished - 2015

Keywords / Materials (for Non-textual outputs)

  • Analgesics, Opioid/chemistry
  • Animals
  • Drug Design
  • Drugs, Investigational/chemistry
  • Humans
  • Narcotic Antagonists/chemistry
  • Nerve Tissue Proteins/agonists
  • Neurons/drug effects
  • Nociception/drug effects
  • Oligopeptides/chemistry
  • Opioid Peptides/chemistry
  • Peptides/chemistry
  • Protein Conformation
  • Protein Engineering
  • Receptors, Opioid/agonists
  • Recombinant Proteins/chemistry


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