A small molecule ligand for the novel pain target, GPR171, produces minimal reward in mice.

ProSAAS is one of the most abundant proteins in the brain and is processed into several smaller peptides. One of which, BigLEN, is an endogenous ligand for the G protein-coupled receptor, GPR171. Recent work in rodent models has shown that a small-molecule ligand for GPR171, MS15203, increases morphine antinociception and is effective in lessening chronic pain. While these studies provide evidence for GPR171 as a possible pain target, its abuse liability has not yet been assessed and was evaluated in the current study. We first mapped the distribution of GPR171 and ProSAAS throughout the reward circuit of the brain using immunohistochemistry and showed that GPR171 and ProSAAS are localized in the hippocampus, basolateral amygdala, nucleus accumbens, prefrontal cortex. In the major dopaminergic structure, the ventral tegmental area (VTA), GPR171 appeared to be primarily localized in dopamine neurons while ProSAAS is outside of dopamine neurons. Next, MS15203 was administered to mice with or without morphine, and VTA slices were stained for the immediate early gene c-Fos as a marker of neuronal activation. Quantification of c-Fos-positive cells revealed no statistical difference between MS15203 and saline, suggesting that MS15203 does not increase VTA activation and dopamine release. The results of a conditioned place preference experiment showed that treatment with MS15203 produced no place preference indicating a lack of reward-related behavior. Taken together this data provides evidence that the novel pain therapeutic, MS15203, has minimal reward liability. Therefore, GPR171 deserves further exploration as a pain target. SIGNIFICANCE STATEMENT: MS15203, a drug that activates the receptor GPR171, was previously shown to increase morphine analgesia. The authors use in vivo and histological techniques to show that it fails to activate the rodent reward circuitry, providing support for the continued exploration of MS15203 as a novel pain drug, and GPR171 a novel pain target.

GPR171 activation regulates morphine tolerance but not withdrawal in a test-dependent manner in mice.

A newly deorphanized G protein-coupled receptor, GPR171, is found to be highly expressed within the periaqueductal gray, a pain-modulating region in the brain. Our recent research has shown that a GPR171 agonist increases morphine antinociception in male mice and opioid signaling in vitro . The objective of this study was to evaluate the effects of combination treatment in females as well as whether chronic treatment can be used without exacerbating morphine-induced tolerance and withdrawal in female and male mice. Our results demonstrate that activation of GPR171 with an agonist attenuates morphine tolerance in both female and male mice on the tail-flick test, but not the hotplate test. Importantly, the GPR171 agonist in combination with morphine does not exacerbate morphine-induced tolerance and withdrawal during long-term morphine treatment. Taken together, these data suggest that the GPR171 agonist may be combined with morphine to maintain antinociception while reducing the dose of morphine and therefore reducing side effects and abuse liability. The outcome of this study is clearly an important step toward understanding the functional interactions between opioid receptors and GPR171 and developing safer therapeutics for long-term pain management.

Morphine-induced kinase activation and localization in the periaqueductal gray of male and female mice.

Morphine is a potent opioid analgesic with high propensity for the development of antinociceptive tolerance. Morphine antinociception and tolerance are partially regulated by the midbrain ventrolateral periaqueductal gray (vlPAG). However, the majority of research evaluating mu-opioid receptor signaling has focused on males. Here, we investigate kinase activation and localization patterns in the vlPAG following acute and chronic morphine treatment in both sexes. Male and female mice developed rapid antinociceptive tolerance to morphine (10 mg/kg i.p.) on the hot plate assay, but tolerance did not develop in males on the tail flick assay. Quantitative fluorescence immunohistochemistry was used to map and evaluate the activation of extracellular signal-regulated kinase 1/2 (ERK 1/2), protein kinase-C (PKC), and protein kinase-A (PKA). We observed significantly greater phosphorylated ERK 1/2 in the vlPAG of chronic morphine-treated animals which co-localized with the endosomal marker, Eea1. We note that pPKC is significantly elevated in the vlPAG of both sexes following chronic morphine treatment. We also observed that although PKA activity is elevated following chronic morphine treatment in both sexes, there is a significant reduction in the nuclear translocation of its phosphorylated substrate. Taken together, this study demonstrates increased activation of ERK 1/2, PKC, and PKA in response to repeated morphine treatment. The study opens avenues to explore the impact of chronic morphine treatment on G-protein signaling and kinase nuclear transport.

GPR171 Agonist Reduces Chronic Neuropathic and Inflammatory Pain in Male, But Not Female Mice.

Chronic pain is a growing public health crisis that requires exigent and efficacious therapeutics. GPR171 is a promising therapeutic target that is widely expressed through the brain, including within the descending pain modulatory regions. Here, we explore the therapeutic potential of the GPR171 agonist, MS15203, in its ability to alleviate chronic pain in male and female mice using a once-daily systemic dose (10 mg/kg, i.p.) of MS15203 over the course of 5 days. We found that in our models of Complete Freund's Adjuvant (CFA)-induced inflammatory pain and chemotherapy-induced peripheral neuropathy (CIPN), MS15203 did not alleviate thermal hypersensitivity and allodynia, respectively, in female mice. On the other hand, MS15203 treatment decreased the duration of thermal hypersensitivity in CFA-treated male mice following 3 days of once-daily administration. MS15203 treatment also produced an improvement in allodynia in male mice, but not female mice, in neuropathic pain after 5 days of treatment. Gene expression of GPR171 and that of its endogenous ligand BigLEN, encoded by the gene PCSK1N, were unaltered within the periaqueductal gray (PAG) in both male and female mice following inflammatory and neuropathic pain. However, following neuropathic pain in male mice, the protein levels of GPR171 were decreased in the PAG. Treatment with MS15203 then rescued the protein levels of GPR171 in the PAG of these mice. Taken together, our results identify GPR171 as a GPCR that displays sexual dimorphism in alleviation of chronic pain. Further, our results suggest that GPR171 and MS15203 have demonstrable therapeutic potential in the treatment of chronic pain.

Opioid-Induced Signaling and Antinociception Are Modulated by the Recently Deorphanized Receptor, GPR171.

ProSAAS is one of the most widely expressed proteins throughout the brain and was recently found to be upregulated in chronic fibromyalgia patients. BigLEN is a neuropeptide that is derived from ProSAAS and was recently discovered to be the endogenous ligand for the orphan G protein-coupled receptor GPR171. Although BigLEN-GPR171 has been found to play a role in feeding and anxiety behaviors, it has not yet been explored in pain and opioid modulation. The purpose of this study was to evaluate this novel neuropeptide-receptor system in opioid-induced antinociception. We found that GPR171 is expressed in GABAergic neurons within the periaqueductal gray, which is a key brain area involved in pain modulation and opioid functions. We also found that, although the GPR171 agonist and antagonist do not have nociceptive effects on their own, they oppositely regulate morphine-induced antinociception with the agonist enhancing and antagonist reducing antinociception. Lastly, we showed that the GPR171 antagonist or receptor knockdown decreases signaling by the mu-opioid receptor, but not the delta-opioid receptor. Taken together, these results suggest that antagonism of the GPR171 receptor reduces mu opioid receptor signaling and morphine-induced antinociception, whereas the GPR171 agonist enhances morphine antinociception, suggesting that GPR171 may be a novel target toward the development of pain therapeutics. SIGNIFICANCE STATEMENT: GPR171 is a recently deorphanized receptor that is expressed within the periaqueductal gray and can regulate mu opioid receptor signaling and antinociception. This research may contribute to the development of new therapeutics to treat pain.