Background & Aims
Current treatment options for chronic pain are inadequate and are often plagued with serious and life-threatening side effects. The opioid epidemic highlights the need for improved therapies. The calcitonin-like receptor (CLR) is a pro-nociceptive G protein-coupled receptor that is a target for migraine pain1. Upon activation by calcitonin gene-related peptide (CGRP), CLR redistributes to endosomes, generating sustained signals that control gene expression involved in pain transmission1,2. However, the kinetics of receptor-specific CLR signaling within intracellular compartments are not well understood. Here, we aimed to examine whether endosomal signaling CLR can be inhibited using targeted nanoparticles as a pain therapeutic.
Methods
We developed a split nanoluciferase (NanoBiT-BRET) assay to investigate agonist-induced assembly of CLR signalosomes at the plasma membrane and in endosomes in HEK293 and human Schwann cells. Hydrophobic ion-pairing (HIP) nanoparticles were loaded with CLR antagonist, olcegepant, and then characterized by the size, charge, and shape, through dynamic light scattering and transmission electron microscopy. Quantum dot (QD)-labelled HIP NPs were used to monitor the uptake of the nanoparticles. Olcegepant-loaded HIP nanoparticles were then tested in NanoBiT-BRET assays for their ability to inhibit CLR signaling.
Results
CGRP induced assembly of CLR, miniG?, and ? arrestin signaling complexes (“signalosomes”) at the plasma membrane, early endosomes, late endosomes, recycling endosomes, and the Golgi apparatus in HEK293 cells. CGRP also induced CLR/miniG?s signalosomes at the plasma membrane, endosomal compartments, and Golgi apparatus in human Schwann cells. Hypertonic sucrose delayed the formation of these CLR signalosomes. HIP nanoparticles loaded with olcegepant were spherical in shape, had high olcegepant loading, had 100 nm hydrodynamic diameter, and exhibited a neutral charge. QD-labelled nanoparticles colocalized with endosomes in 2 to 4 hours. HIP nanoparticles loaded with olcegepant inhibited CLR/miniG?s complexes at the plasma membrane, endosomes, while empty HIP particles did not affect complex formation at the plasma membrane or in subcellular organelles.
Conclusions
CLR signalosomes were formed at the plasma membrane and subcellular compartments when activated by CGRP. CLR antagonist loaded into HIP nanoparticles prevented CLR signalosome formation. Thus, nanoparticles may be a superior approach to deliver CLR antagonists for the treatment of pain.
References
1.Al-Hassany L, Goadsby PJ, Danser AHJ, MaassenVanDenBrink A. Calcitonin gene-related peptide-targeting drugs for migraine: how pharmacology might inform treatment decisions. Lancet Neurol. 2022 Mar;21(3):284-294. doi: 10.1016/S1474-4422(21)00409-9. Epub 2022 Jan 31. Erratum in: Lancet Neurol. 2022 Oct; 21(10): e9.
2.Yarwood RE, Imlach WL, Lieu T, Veldhuis NA, Jensen DD, Klein Herenbrink C, Aurelio L, Cai Z, Christie MJ, Poole DP, Porter CJH, McLean P, Hicks GA, Geppetti P, Halls ML, Canals M, Bunnett NW. Endosomal signaling of the receptor for calcitonin gene-related peptide mediates pain transmission. Proc Natl Acad Sci U S A. 2017 Nov 14; 114(46): 12309-12314.
Presenting Author
Shavonne Teng
Poster Authors
Topics
- Mechanisms: Biological-Molecular and Cell Biology