Targeting of subcellular metabotropic glutamate receptor 5 signaling to modulate pain transmission

Abstract Metabotropic glutamate receptor 5 (mGlu 5 ) is a class C GPCR crucial for neuronal development and synaptic transmission. mGlu 5 is a potential therapeutic target in pain management and modulates pain-associated gene expression and signaling pathways. Although mGlu 5 inhibitors have shown promise in treating pain, none have translated to the clinic. Up to 90% of neuronal mGlu 5 expression is intracellular, although the precise locations and function of different mGlu 5 intracellular pools remains unclear. Building on recent evidence showing the importance of endosome-mediated nociceptive signaling by other GPCRs, we hypothesized that endosomal pools of mGlu 5 contribute to pain transmission, and that targeted inhibition of intracellular mGlu 5 signaling results in superior analgesia. Using calcium mobilization assays and genetically encoded resonance energy transfer biosensors, we report that upon its activation mGlu 5 recruits Gα q/11 and Gα s to the plasma membrane. Conversely, internalized mGlu 5 in endosomes recruits only Gα q/11 proteins. mGlu 5 signaling is highly dependent on receptor trafficking to endosomes, with sustained nuclear ERK1/2 signaling requiring both receptor internalization and active glutamate transport into the cell. We generated pH responsive nanoparticles loaded with the mGlu 5 negative allosteric modulator VU0366058 (DIPMA-VU058), enabling endosome-targeted inhibition of mGlu 5 . Nanoparticle encapsulation of VU0366058 enhanced inhibition of both acute and sustained nuclear ERK1/2 signaling, and significantly reduced neuronal excitability in nociceptive circuits in spinal cord slices from rats with neuropathic pain. Intrathecal administration of DIPMA-VU058 achieved superior analgesia in both inflammatory and neuropathic models of pain in mice compared to free VU0366058 and the reference compound fenobam. These studies demonstrate the importance of endosome-associated receptors for the complete mGlu 5 signaling response. Furthermore, we show that manipulating the cellular distribution of an allosteric modulator can engender location-biased pharmacological effects. Together, we have revealed new and unappreciated roles for endosome-specific mGlu5 signaling and demonstrate that endosome-selective targeting may offer an alternative therapeutic approach for modulating mGlu 5 activity.