Design of a Stable Cyclic Peptide Analgesic Derived from Sunflower Seeds that Targets the κ-Opioid Receptor for the Treatment of Chronic Abdominal Pain

Abstract

The rising opioid crisis has become a worldwide societal and public health burden, resulting from the abuse of prescription opioids. Targeting the κ-opioid receptor (KOR) in the periphery has emerged as a powerful approach to develop novel pain medications without central side effects. Inspired by the traditional use of sunflower ( Helianthus annuus ) preparations for analgesic purposes, we developed novel stabilized KOR ligands (termed as helianorphins) by incorporating different dynorphin A sequence fragments into a cyclic sunflower peptide scaffold. As a result, helianorphin-19 selectively bound to and fully activated the KOR with nanomolar potency. Importantly, helianorphin-19 exhibited strong KOR-specific peripheral analgesic activity in a mouse model of chronic visceral pain, without inducing unwanted central effects on motor coordination/sedation. Our study provides a proof of principle that cyclic peptides from plants may be used as templates to develop potent and stable peptide analgesics applicable via enteric administration by targeting the peripheral KOR for the treatment of chronic abdominal pain.

Genome Mining-Based Discovery of Blenny Fish-Derived Peptides Targeting the Mouse κ-Opioid Receptor

“…On the other hand, all three blenny fish-derived peptides demonstrated high potency and efficacy in the cAMP assay. Previous studies identified the arginine residues at the position 6 and 7 of human dyn A 1-13 to be crucial for its high a Binding and cAMP data for dynorphin (dyn) A 1-13 have been taken from previously published literature (Muratspahić et al, 2021). b Data are from three independent experiments.…”

“…All blenniorphins comprise these residues in their sequences. Hence, it is thus not surprising that they stimulate a mammalian KOR with a potency comparable to human dyn A 1-13 (Muratspahić et al, 2021). However, the rank order potency for the blenniorphans in binding vs. functional assays does not match.…”

“…Cell culture, transfection and cloning of mouse KOR have been performed as previously described (Muratspahić et al, 2021).…”

“…HEK293 cell membranes stably expressing mouse KOR were prepared as previously described (Nasrollahi-Shirazi et al, 2020) and were used in radioligand displacement binding assays as described in Muratspahić et al (Muratspahić et al, 2021) Briefly, the assay was carried out in duplicates in a final volume of 300 µl containing binding buffer (50 mM Tris-HCl pH 7.4, 10 mM MgCl 2 , and 0.1% bovine serum albumin), [ 3 H]-diprenorphine (1 nM final concentration), peptides (logarithmically spaced concentrations covering the range of 0.1 nM-30 µM) and membranes (6.3-7.6 µg/assay). The reaction mixture was incubated for 1.5 h at 37 °C.…”

“…IC 50 -values were calculated by fitting the data obtained in radioligand displacement binding assays to a three-parameter logistic Hill equation and converted to inhibition constants (K i ) using the Cheng and Prusoff approximation. K d (0.87 ± 0.06 nM) and Bmax (7,166 ± 147 fmol/mg protein) values of [ 3 H]-diprenorphine were previously determined (Muratspahić et al, 2021). Data were expressed in counts per min (cpm) of total binding of the radioligand, whereas 1 cpm a 38.4 fmoles.…”