A lipid-anchored neurokinin 1 receptor antagonist prolongs pain relief by a three-pronged mechanism of action targeting the receptor at the plasma membrane and in endosomes

Quynh N Mai,Priyank Shenoy,Tim Quach,Jeffri S Retamal,Arisbel B Gondin,Holly R Yeatman,Luigi Aurelio,Joshua W Conner,Daniel P Poole,Meritxell Canals,Cameron J Nowell,Bim Graham,Thomas P Davis,Stephen J Briddon,Stephen J Hill,Christopher J.H Porter,Nigel W Bunnett,Michelle L Halls,Nicholas A Veldhuis

doi:10.1016/j.jbc.2021.100345

Abstract

G-protein-coupled receptors (GPCRs) are traditionally known for signaling at the plasma membrane, but they can also signal from endosomes after internalization to control important pathophysiological processes. In spinal neurons, sustained endosomal signaling of the neurokinin 1 receptor (NK1R) mediates nociception, as demonstrated in models of acute and neuropathic pain. An NK1R antagonist, Spantide I (Span), conjugated to cholestanol (Span-Chol), accumulates in endosomes, inhibits endosomal NK1R signaling, and causes prolonged antinociception. However, the extent to which the Chol-anchor influences long-term location and activity is poorly understood. Herein, we used fluorescent correlation spectroscopy and targeted biosensors to characterize Span-Chol over time. The Chol-anchor increased local concentration of probe at the plasma membrane. Over time we observed an increase in NK1R-binding affinity and more potent inhibition of NK1R-mediated calcium signaling. Span-Chol, but not Span, caused a persistent decrease in NK1R recruitment of β-arrestin and receptor internalization to early endosomes. Using targeted biosensors, we mapped the relative inhibition of NK1R signaling as the receptor moved into the cell. Span selectively inhibited cell surface signaling, whereas Span-Chol partitioned into endosomal membranes and blocked endosomal signaling. In a preclinical model of pain, Span-Chol caused prolonged antinociception (>9 h), which is attributable to a three-pronged mechanism of action: increased local concentration at membranes, a prolonged decrease in NK1R endocytosis, and persistent inhibition of signaling from endosomes. Identifying the mechanisms that contribute to the increased preclinical efficacy of lipid-anchored NK1R antagonists is an important step toward understanding how we can effectively target intracellular GPCRs in disease

Highlights

G-protein-coupled receptors (GPCRs) are traditionally known for signaling at the plasma membrane, but they can signal from endosomes after internalization to control important pathophysiological processes
Lipid anchors increase the available concentration of drug at the cell surface Inspired by prior studies using lipid–drug conjugates [18, 19], we previously synthesized a series of lipid-anchored probes comprising the sterol cholestanol as a lipid conjugate for anchoring a cargo to membranes via a flexible polyethylene glycol linker (PEG4-PEG3-PEG4) [5]
Fluorescence correlation spectroscopy (FCS) enables measurement of the concentrations of fluorescent molecules within a small defined volume (

Summary

Introduction

G-protein-coupled receptors (GPCRs) are traditionally known for signaling at the plasma membrane, but they can signal from endosomes after internalization to control important pathophysiological processes. In a preclinical model of pain, Span-Chol caused prolonged antinociception (>9 h), which is attributable to a threepronged mechanism of action: increased local concentration at membranes, a prolonged decrease in NK1R endocytosis, and persistent inhibition of signaling from endosomes. This includes pHresponsive nanoparticles that deliver and release the NK1R antagonist aprepitant directly into the endosomes [17] and lipid-anchored NK1R antagonists that accumulate in endosomal membranes [5] Both of these approaches improved drug efficacy in preclinical models of pain (2–5-fold more effective antinociception, 2–4-fold longer duration of action compared with free drug) [5, 17]. It is possible that lipid-anchored antagonists affect the signaling and trafficking of plasma membrane-localized NK1R, in addition to their later antagonism of endosomal receptors. This dual antagonism—initial blockade of plasma membrane receptors during partitioning into the plasma membrane and prolonged blockade of the pathophysiologically relevant signal from endosomes—could enhance therapeutic efficacy

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