The sensation and transmission of pain are essential physiological processes that allow us to detect and react to harmful stimuli and initiate inflammatory responses to protect damaged tissue and promote wound healing. Peripheral and central processes that lead to pain transmission are highly adaptive, and the pain experienced is usually proportional to the extent of the injury. As a part of this adaptive physiological response, a heightened sensitivity to pain occurs to provide awareness of damaged tissue and maintain protective behavior for the duration of an injury.

As healing occurs, this sensitization typically reduces over time. In contrast, in chronic inflammatory and neuropathic pain conditions such as arthritis, fibromyalgia or diabetic-related neuropathy, where damaged tissue is unable to heal or inflammatory mediators continue to be produced, this sensitization fails to diminish and can cause significant discomfort and loss of function over extended time periods (Scholz and Woolf, 2002). This is typically described through two phenomena: (a) allodynia, where one feels pain in response to a normally non-painful stimulus; and (b) hyperalgesia, where one experiences an exacerbated pain sensation to a moderately painful stimulus (Baron, 2006; Steeds, 2016). Due to the complexity of chronic pain and significant limitations with safety and compliance for available analgesics, these conditions are extremely difficult to manage, thus impacting the quality of life for many patients.

Despite many advances in basic research and in the clinic, the analgesic landscape in recent decades has seen few changes, due to the limited availability of effective analgesic agents and the potential for abuse of routinely prescribed drugs (Dowell et al., 2016; Goodman and Brett, 2017). In the midst of a growing opioid crisis (Schuchat et al., 2017), the development of new pain medicines is becoming increasingly important. For safety and logistical reasons, the most obvious gains can be made by repurposing Food and Drug Administration (FDA)-approved drugs that are currently used for other indications (e.g., anti-depressants; Kremer et al., 2016; Cooper et al., 2017) or re-formulating established analgesics such as opioids to improve pharmacokinetic profiles (Saraghi and Hersh, 2013). However, new and effective therapeutic approaches may also be gained through greater characterization of the underlying cellular and molecular mechanisms that lead to pain, as a means to identify new molecular targets and further define how analgesic side-effects occur and can be avoided.

G protein-coupled receptors (GPCRs) are important mediators of pain or analgesia and many of these receptors participate in dynamic trafficking processes such as endocytosis, as a part of their activity cycle. It is now evident that receptor trafficking is also critical for the initiation of spatially and temporally distinct signaling events, and importantly, some of these location-specific or compartmentalized processes are associated with greater modulation of pain (Geppetti et al., 2015; Irannejad et al., 2017; Stoeber et al., 2018; Thomsen et al., 2018). Here, we address limitations of the current analgesic landscape and look to new drug discovery studies focused on GPCRs that participate in dynamic trafficking processes in neurons. New biophysical tools that have been used to characterize compartmentalized signaling reveal how the membrane partitioning properties of drugs influence their functional selectivity for location-specific processes. This knowledge has been exploited through the use of lipid-anchored drug conjugates that increase GPCR targeting in specific subcellular domains, to enhance analgesic outcomes through the inhibition of endosomal signaling.

Chronic or persistent pain incorporates a complex range of disorders that requires a combination of non-pharmacological and pharmacological approaches for treatment. From a pharmacological perspective, treatment is possible by administering one or more therapeutic agents such as paracetamol/acetaminophen, non-steroidal anti-inflammatory drugs (NSAIDs) or cyclooxygenase-2 inhibitors (Coxibs) followed by careful use of opioids for elevated pain (e.g., morphine or oxycodone). Unfortunately, each of these drugs has associated side-effects that limit their use. NSAIDs and Coxibs have potential cardiovascular and gastrointestinal side effects (Whelton, 2000), and should be used more sparingly than paracetamol/acetaminophen, which carries a risk of hepatotoxicity with excessive use (Mahadevan et al., 2006). While opioids remain some of the most effective analgesics available in the clinic, they have a high abuse potential due to their euphoric or addictive properties, and where repeated use leads to receptor desensitization and tolerance. To overcome tolerance, patients with chronic pain can be subjected to sustained increases in dosing or switching to other more potent opioids to improve analgesia, which often provides only temporary gains in pain relief. However, this approach may increase the risk of tolerance and addiction over time, in addition to increasing the likelihood of debilitating side-effects such as constipation and respiratory depression (Corbett et al., 2006; Boudreau et al., 2009; Volkow et al., 2011).

Alternative GPCR targets have been identified to reduce reliance on opioid analgesics. Cannabinoids, which are proposed as effective opioid alternatives, reduce pain through activation of G_i/o-coupled cannabinoid receptors (primarily CB₁), which leads to the downregulation of excitatory processes, and modulation of serotoninergic (5-HT) and noradrenergic pathways. Although widely available and used for millennia, we are yet to see the outcomes of systematic use in the clinic for treating pain, and it is also acknowledged to lead to behavioral risks that require further investigation (Mendiguren et al., 2018). Gabapentinoids such as gabapentin or pregabalin, target the α2δ subunit of voltage-gated calcium channels and have been approved as first-line medications to manage neuropathic pain (e.g., postherpetic neuralgia, fibromyalgia). These were initially used for the treatment of epilepsy, and in some cases for anxiety disorders. Although regarded as relatively safe drugs, safety concerns for gabapentinoids have grown and include excessive usage and behavioral risks such as suicidal behavior (Johansen, 2018; Molero et al., 2019). Together, this provides a small insight into established and emerging risks associated with common analgesics. This raises the question of whether any of these compounds can be modified to improve their safety profiles and if new or emerging targets are available. We discuss these points below in the context of receptor trafficking, which is a critical component of the activity cycle for many molecular pain targets.

Members of the GPCR superfamily are considered to be druggable targets due to high levels of cell surface expression and their ability to contribute to all pathophysiological processes, including pain. Accordingly, GPCR-selective drugs represent more than one-third of all FDA-approved medicines (Hauser et al., 2017). There are at least 40 members of the GPCR family that are considered to be potential therapeutic targets for the regulation of pain (Stone and Molliver, 2009). Yet despite advanced drug discovery programs for multiple receptors, and abuse concerns for opioid receptors, very few targets have clinically succeeded beyond opioids in the past decade, with notable exceptions being the recent approval of Fremanezumab, Eptinezumab, Galcanezumab, and Erenumab for treatment of migraine, being monoclonal antibodies that target the neuropeptide calcitonin gene-related peptide (CGRP) or its receptor, Calcitonin Receptor-Like Receptor/Receptor Activity-Modifying Protein 1 (CLR/RAMP1; see review by Scuteri et al., 2019).

There are a number of challenges in the early phase of analgesic drug discovery for GPCRs. This includes safety concerns for targets that have overlapping functions in other tissues, and inaccurate evaluations of efficacy when using relatively simplified rodent-based pre-clinical pain models to represent the complexity of clinical pain conditions or characterize human-selective compounds (Mao, 2012). Furthermore, the localization of receptors in pre-synaptic and post-synaptic neurons is critical for the activity cycle and nociceptive outputs of several GPCRs (Figure 1). On a cellular level, considerations for the intracellular disposition of analgesics and their ability to regulate receptor trafficking and localization have also recently been proposed to be an important part of the drug discovery process (Jensen et al., 2017; Yarwood et al., 2017; Jimenez-Vargas et al., 2018; Stoeber et al., 2018).

GPCRs are highly dynamic proteins that achieve distinct signaling outcomes by adopting different conformational states (Rasmussen et al., 2011; Latorraca et al., 2017). Extracellular ligands that bind cell surface GPCRs promote receptor conformations that activate heterotrimeric G proteins to transduce downstream signaling and also favor phosphorylation by GPCR kinases (GRKs). This phosphorylation occurs primarily at the C-terminus to enhance engagement with β-arrestins (βArrs), which can function as adaptor proteins to mediate distinct signaling processes such as MAPK activity, and also facilitate interactions with clathrin-coated membranes to promote endocytosis into endosomes (Ferguson et al., 1996). This was historically considered to facilitate termination of signaling by targeting receptors to degradative pathways, or rapid receptor recycling to reset the activity cycle during the internalization process, and increase the potential for sustained signaling once the receptor is recovered at the plasma membrane (PM; Ferguson et al., 1996; Shukla et al., 2014).

A more recent theory has emerged, suggesting that a third trafficking possibility exists, whereby receptors can remain on intracellular membranes such as endosomes for sustained periods of time, to facilitate distinct signaling processes in a βArr- or a G protein-dependent manner. This paradigm shift was initially revealed by studies on Gs-coupled receptors such as the parathyroid (PTHR), thyroid-stimulating hormone (TSHR) and β2 adrenergic receptors to demonstrate that endosomal-mediated sustained cyclic adenosine monophosphate (cAMP) production could be observed after endocytosis has occurred (reviewed in detail by Vilardaga et al., 2014; Tsvetanova et al., 2015; Thomsen et al., 2018).

The development of genetically encoded tools such as conformation-selective nanobodies, Förster/Fluorescence Resonance Energy Transfer (FRET) or Bioluminescence Resonance Energy Transfer (BRET) biosensors, have provide highly sensitive approaches for observing and measuring dynamic activation states and spatiotemporal signaling [e.g., compartmentalized cAMP production, Protein kinase C (PKC) and Extracellular signal-regulated kinases (ERK) activity] of GPCRs in real-time (Irannejad et al., 2017; Halls and Canals, 2018). Given the prevalence and importance of trafficking GPCRs in neurons, the internalization and location-specific signaling of several GPCRs with established roles in pain have been described, including but not limited to the Neurokinin 1 Receptor (NK₁R), CLR/RAMP1, metabotropic glutamate receptor 5 (mGluR5), chemokine receptor (CCR1), Protease-Activated Receptor 2 (PAR₂) and the Mu Opioid Receptor (MOR; Mantyh et al., 1995a; O’Malley et al., 2003; Gilliland et al., 2013; Poole et al., 2015; Jensen et al., 2017; Yarwood et al., 2017; Stoeber et al., 2018). An overview of these trafficking outcomes is summarized in Table 1, to reveal how stimulation with endogenous ligands alters receptor localization in vitro, or in pre-clinical pain models.

More recently, conformation-selective single-domain camelid antibodies (nanobodies) that can detect and bind active-state receptors have been instrumental for advancing this concept to other organelles. Distinct nanobody clones that are known to engage with the β₁ Adrenergic Receptor (β₁AR) or MOR have been shown to be recruited to the Golgi apparatus in a GPCR activity-dependent manner independently from initial stimulation at the cell surface. Specifically, this is achieved using relatively lipophilic ligands that can freely diffuse throughout the cell, or hydrophilic compounds that are proposed to access Golgi pools via transporters (Irannejad et al., 2017; Stoeber et al., 2018).

These important pharmacological insights have significant implications for understanding how drugs may exert their effects (or side-effects) and are consistent with other receptors that contribute to pain transmission. For example, endogenous peptide-based enkephalins can stimulate MOR and Delta-Opioid Receptor (DOR) to activate rapid signaling processes in micro-domains of the cell surface and sustained signaling from endosomes (Finn and Whistler, 2001; Groer et al., 2011; Halls et al., 2016; Stoeber et al., 2018), whereas non-peptide opioids such as morphine can freely diffuse through cells to stimulate Golgi pools of the MOR, and initiate a spatiotemporally distinct wave of signaling. The importance of opioid-induced Golgi signaling for analgesia and its association with safety outcomes remains to be determined in vivo (Stoeber et al., 2018).

Under pathological pain conditions, the excitatory mGluR5 has been detected in intracellular locations, including the inner nuclear membrane and endoplasmic reticulum (ER; Jong et al., 2014; Purgert et al., 2014; Vincent et al., 2016, 2017). Stimulated mGluR5 couples with Gα_q to evoke cytoplasmic and nuclear calcium mobilization (Jong et al., 2009). Furthermore, in models of spared-nerve injury (Vincent et al., 2016) and inflammatory pain (Vincent et al., 2017), 60% of the mGluR5 receptor population was shown to be localized to the inner nuclear membrane in spinal dorsal horn neurons (Vincent et al., 2016). Importantly, activation of nuclear mGluR5 leads to sustained nuclear Ca²⁺ signaling, phosphorylation of ERK1/2 and induction of c-fos expression, leading to increased nociceptive hypersensitivity (Lee et al., 2008; Jong et al., 2009; Purgert et al., 2014; Vincent et al., 2016, 2017). Blockade of cell surface mGluR5 by the impermeable antagonist LY393053 resulted in limited analgesia and modest reductions in second messenger coupling. In contrast, the membrane-permeable antagonist fenobam significantly reduced mechanical allodynia, MAP kinase (ERK1/2) phosphorylation and c-fos expression in a spared-nerve injury pain model. Although these differences may be caused by a range of factors including drug disposition and differences in potencies, it may also provide indirect evidence for the initiation of distinct mGluR5-dependent pain responses from different cellular locations (Lax et al., 2014; Vincent et al., 2016, 2017). Focused drug discovery around cell-permeant compounds biased toward intracellular mGluR5 pools is warranted and may lead to new opportunities for targeting glutamate signaling for analgesia.

The studies above suggest that GPCRs that undergo endocytosis may be modulated more effectively by ligands that can diffuse to intracellular sites. This raises questions about whether the intrinsic properties of analgesic agents can be enhanced by chemical modification, to increase activity or partitioning into membranes where GPCRs are known to initiate signals associated with pain.

The signaling and trafficking of GPCRs is important for mediating physiological processes at the PM and can also drive distinct, compartmentalized signaling events from intracellular sites. In the context of pain, defining this relationship may provide significant opportunities for neuropharmacology and analgesic drug discovery. However, while this may provide important insights that pinpoint discrete signaling outcomes most closely associated with modulating pain behaviors, or favorable drug properties that achieve analgesia while avoiding safety issues, it also critical to translate these proof of concept studies to human tissues and diseases. It remains unknown (and very challenging), for example, to demonstrate how the Golgi-specific MOR-signaling component influences analgesia or other side-effects in animals or humans, or if pH-sensitive fentanyl analogs provide genuine advantages over the parent compound in humans with chronic pain.

Although a relatively new phenomenon, ligands that have been identified or modified to possess unique location-biased properties have provided both interesting and valuable proof of concept findings that warrant further investigation. This includes receptors discussed in this review article and many others that contribute to pain in both neurons and non-neuronal cells that drive signaling processes that lead to sustained pain. With the availability of powerful new technologies and biophysical tools, it is predicted that further in-depth compartmentalized signaling-focused drug discovery studies on other trafficking receptors will provide many more valuable insights and other location-specific drug targets.

JR, PR-G, PS, DP and NV wrote the manuscript.

Research in NV and DP laboratories is funded in part by Takeda Pharmaceuticals Inc.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.