G protein-coupled receptors (GPCRs) are dynamic cell surface receptors that control most physiological processes by initiating intracellular signalling pathways that are both spatially and temporally controlled. GPCRs are conventionally thought to bind ligands and initiate signalling pathways from the cell surface, and this signalling can be subsequently “arrested” via GPCR interactions with β-arrestin adaptor proteins that recruit receptors into the endosomal network and away from the cell surface. However, new evidence suggests that GPCR internalisation is not only important for switching off cell surface signalling, but that, endosomes are now considered a platform for generating spatially and temporally distinct signalling outputs. Importantly, CBNS researchers demonstrated that GPCR-mediated “endosomal signalling” in spinal neurons is associated with pathophysiological processes such as sustained pain transmission.
The selective delivery of GPCR inhibitors directly to endosomes is a unique and novel strategy for effective and selective inhibition of chronic pain or other conditions. Two endosomal delivery strategies are currently being investigated: A) Conjugation of inhibitors to a lipid anchor (cholestanol) to promote endosomal targeting and retention; B) pH-sensitive soft nanoparticles for delivery of antagonists into endosomes.
Using real- time fluorescence confocal microscopy and pharmacological analyses in cells and animals, our data demonstrates that cholestanol conjugates can bind GPCRs at the cell surface and in endosomes, and are very effective at selectively blocking endosomal signals thereby providing long-term analgesia that is not observed with a non-lipidated drug equivalent (manuscript under review). Importantly, in 2016 these findings were extended to multiple cholestanol conjugates that target different GPCRs, to demonstrate that endosomal inhibition could be applied to many trafficking proteins.
pH-responsive nanoparticles: Developing a nanoparticle that carefully “tunes” cell signalling without affecting cell viability poses a significant challenge. In particular, cytotoxicity can be caused by protonation of pH- responsive monomers in the hydrophobic portion of the diblock copolymer when particles disassemble within the acidic environment of early and late endosomes. To reduce this toxicity, we have incorporated non-charged monomers units by RAFT to “shield” the cell from protonated polymers.
Fluorescence microscopy has confirmed these particles remain within endosomes (i.e. do not promote endosomal escape). Most importantly, for the first time we have observed improved analgesia with spinal administration of drug- loaded nanoparticles when compared to the drug alone or empty particles. These studies are ongoing. Future cell signalling assays will provide a clearer understanding of how these particles are regulating endosomal signalling.