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Substance P (SP) is a highly conserved member of the tachykinin peptide family that is widely expressed throughout the animal kingdom. The numerous members of the tachykinin peptide family are involved in a multitude of neuronal signaling pathways, mediating sensations and emotional responses (Steinhoff et al. in Physiol Rev 94:265–301, 2014). In contrast to receptors for classical transmitters, such as glutamate (Parsons et al. in Handb Exp Pharmacol 249–303, 2005), only a minority of neurons in certain brain areas express neurokinin receptors (NKRs) (Mantyh in J Clin Psychiatry 63:6–10, 2002). SP is also expressed by a variety of non-neuronal cell types such as microglia, as well as immune cells (Mashaghi et al. in Cell Mol Life Sci 73:4249–4264, 2016). SP is an 11-amino acid neuropeptide that preferentially activates the neurokinin-1 receptor (NK1R). It transmits nociceptive signals via primary afferent fibers to spinal and brainstem second-order neurons (Cao et al. in Nature 392:390–394, 1998). Compounds that inhibit SP’s action are being investigated as potential drugs to relieve pain. More recently, SP and NKR have gained attention for their role in complex psychiatric processes. It is a key goal in the field of pain research to understand mechanisms involved in the transition between acute pain and chronic pain. The influence of emotional and cognitive inputs and feedbacks from different brain areas makes pain not only a perception but an experience (Zieglgänsberger et al. in CNS Spectr 10:298–308, 2005; Trenkwaldner et al. Sleep Med 31:78–85, 2017). This review focuses on functional neuronal plasticity in spinal dorsal horn neurons as a major relay for nociceptive information.

The neuropeptide substance P (SP) is important in pain and inflammation. SP activates the neurokinin-1 receptor (NK1R) to signal via Gq and Gs proteins. Neurokinin A also activates NK1R, but leads to selective Gq signaling. How two stimuli yield distinct G protein signaling at the same G protein-coupled receptor remains unclear. We determined cryogenic-electron microscopy structures of active NK1R bound to SP or the Gq-biased peptide SP6–11. Peptide interactions deep within NK1R are critical for receptor activation. Conversely, interactions between SP and NK1R extracellular loops are required for potent Gs signaling but not Gq signaling. Molecular dynamics simulations showed that these superficial contacts restrict SP flexibility. SP6–11, which lacks these interactions, is dynamic while bound to NK1R. Structural dynamics of NK1R agonists therefore depend on interactions with the receptor extracellular loops and regulate G protein signaling selectivity. Similar interactions between other neuropeptides and their cognate receptors may tune intracellular signaling.Determination of the cryo-EM structures of active neurokinin-1 receptor bound to substance P or the Gq biased peptide SP6–11 reveals that interactions with the receptor extracellular loops regulate G protein signaling selectivity.

The hypothesis that sustained G protein-coupled receptor (GPCR) signaling from endosomes mediates pain is based on studies with endocytosis inhibitors and lipid-conjugated or nanoparticle-encapsulated antagonists targeted to endosomes. GPCR antagonists that reverse sustained endosomal signaling and nociception are needed. However, the criteria for rational design of such compounds are ill-defined. Moreover, the role of natural GPCR variants, which exhibit aberrant signaling and endosomal trafficking, in maintaining pain is unknown. Herein, substance P (SP) was found to evoke clathrin-mediated assembly of endosomal signaling complexes comprising neurokinin 1 receptor (NK₁R), Gαq/i, and βarrestin-2. Whereas the FDA-approved NK₁R antagonist aprepitant induced a transient disruption of endosomal signals, analogs of netupitant designed to penetrate membranes and persist in acidic endosomes through altered lipophilicity and pKa caused sustained inhibition of endosomal signals. When injected intrathecally to target spinal NK₁R+ve neurons in knockin mice expressing human NK₁R, aprepitant transiently inhibited nociceptive responses to intraplantar injection of capsaicin. Conversely, netupitant analogs had more potent, efficacious, and sustained antinociceptive effects. Mice expressing C-terminally truncated human NK₁R, corresponding to a natural variant with aberrant signaling and trafficking, displayed attenuated SP-evoked excitation of spinal neurons and blunted nociceptive responses to SP. Thus, sustained antagonism of the NK₁R in endosomes correlates with long-lasting antinociception, and domains within the C-terminus of the NK₁R are necessary for the full pronociceptive actions of SP. The results support the hypothesis that endosomal signaling of GPCRs mediates nociception and provides insight into strategies for antagonizing GPCRs in intracellular locations for the treatment of diverse diseases.

Nanoparticle-mediated drug delivery is especially useful for targets within endosomes because of the endosomal transport mechanisms of many nanomedicines within cells. Here, we report the design of a pH-responsive, soft polymeric nanoparticle for the targeting of acidified endosomes to precisely inhibit endosomal signalling events leading to chronic pain. In chronic pain, the substance P (SP) neurokinin 1 receptor (NK1R) redistributes from the plasma membrane to acidified endosomes, where it signals to maintain pain. Therefore, the NK1R in endosomes provides an important target for pain relief. The pH-responsive nanoparticles enter cells by clathrin- and dynamin-dependent endocytosis and accumulate in NK1R-containing endosomes. Following intrathecal injection into rodents, the nanoparticles, containing the FDA-approved NK1R antagonist aprepitant, inhibit SP-induced activation of spinal neurons and thus prevent pain transmission. Treatment with the nanoparticles leads to complete and persistent relief from nociceptive, inflammatory and neuropathic nociception and offers a much-needed non-opioid treatment option for chronic pain.

Clostridioides difficile infection (CDI) is a major cause of healthcare-associated gastrointestinal infections . The exaggerated colonic inflammation caused by C. difficile toxins such as toxin B (TcdB) damages tissues and promotes C. difficile colonization , but how TcdB causes inflammation is unclear. Here we report that TcdB induces neurogenic inflammation by targeting gut-innervating afferent neurons and pericytes through receptors, including the Frizzled receptors (FZD1, FZD2 and FZD7) in neurons and chondroitin sulfate proteoglycan 4 (CSPG4) in pericytes. TcdB stimulates the secretion of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) from neurons and pro-inflammatory cytokines from pericytes. Targeted delivery of the TcdB enzymatic domain, through fusion with a detoxified diphtheria toxin, into peptidergic sensory neurons that express exogeneous diphtheria toxin receptor (an approach we term toxogenetics) is sufficient to induce neurogenic inflammation and recapitulates major colonic histopathology associated with CDI. Conversely, mice lacking SP, CGRP or the SP receptor (neurokinin 1 receptor) show reduced pathology in both models of caecal TcdB injection and CDI. Blocking SP or CGRP signalling reduces tissue damage and C. difficile burden in mice infected with a standard C. difficile strain or with hypervirulent strains expressing the TcdB2 variant. Thus, targeting neurogenic inflammation provides a host-oriented therapeutic approach for treating CDI.

The peptide substance P (SP) and the cytokine tumor necrosis factor (TNF) have been implicated in inflammatory processes. Mast cells are recognized as important in inflammatory responses. Here, we report that IL-33 (30 ng/mL), a member of the IL-1 family of cytokines, administered in combination with SP (1 μM), markedly increase (by 1,000-fold) TNF gene expression in cultured human LAD2 and primary mast cells derived from umbilical cord blood. SP (0.01–1 μM) and IL-33 (1–100 ng/mL) in combination also greatly stimulate TNF secretion (by 4,500-fold). Pretreatment of LAD2 cells with two different neurokinin-1 (NK-1) receptor antagonists and siRNA inhibits TNF secretion by 50% (P < 0.001) when stimulated by SP and IL-33. Pretreatment of LAD2 cells with a neutralizing antibody for IL-33 receptor, ST2, inhibits TNF secretion by 50% (P < 0.001), and ST2 siRNA decreases TNF secretion by 30% (P < 0.05), when stimulated by SP and IL-33. Surprisingly, NK-1 antagonists also inhibit 50% of TNF secretion (P < 0.001) when stimulated only by IL-33, and ST2 receptor reduction also decreases SP-stimulated TNF secretion by 30% (P < 0.05), suggesting an interaction between NK-1 and ST2 receptors. Moreover, IL-33 increases NK-1 gene and surface protein expression, as well as IKβ-α phosphorylation. Pretreatment of LAD2 cells with 5,7,3′,4′-tetramethoxyflavone (methoxyluteolin) (1–100 μM) inhibits (P < 0.001) TNF gene expression (98%) and secretion (64%) at 50 μM and phosphorylation of p-IKB-α at 1 μM when stimulated by SP and IL-33. These findings identify a unique amplification process of TNF synthesis and secretion via the interaction of NK-1 and ST2 receptors inhibitable by methoxyluteolin.

Unlike conventional itch, neuropathic itch develops in normal skin from excess peripheral firing or dampened central inhibition of itch pathway neurons. Neuropathic itch is a symptom of the same central and peripheral nervous system disorders that cause neuropathic pain, such as sensory polyneuropathy, radiculopathy, herpes zoster, stroke, or multiple sclerosis, and lesion location affects symptoms more than aetiology. The causes of neuropathic itch are heterogeneous, and thus diagnosis is based primarily on recognising characteristic, disease-specific clinical presentations. However, the diagnosis of neuropathic itch is challenging, different subforms exist (eg, focal vs widespread, peripheral vs central), and the mechanisms of neuropathic itch are poorly understood, resulting in reduced treatment availability. Currently available strategies include treating or preventing causal diseases, such as diabetes or herpes zoster, and topical or systemic medications that calm excess neuronal firing. Discovery of itch mediators such as gastrin releasing peptide, receptors (eg, neurokinin-1), and pathways (eg, Janus kinases) might encourage much needed new research into targeted treatments of neuropathic itch.

Comparisons of G protein-coupled receptor (GPCR) complexes with agonists and antagonists based on X-ray crystallography and cryo-electron microscopy structure determinations show differences in the width of the orthosteric ligand binding groove over the range from 0.3 to 2.9 Å. Here, we show that there are transient structure fluctuations with amplitudes up to at least 6 Å. The experiments were performed with the neurokinin 1 receptor (NK1R), a GPCR of class A that is involved in inflammation, pain, and cancer. We used 19F-NMR observation of aprepitant, which is an approved drug that targets NK1R for the treatment of chemotherapy-induced nausea and vomiting. Aprepitant includes a bis-trifluoromethyl-phenyl ring attached with a single bond to the core of the molecule; 19F-NMR revealed 180° flipping motions of this ring about this bond. In the picture emerging from the 19F-NMR data, the GPCR transmembrane helices undergo large-scale floating motions in the lipid bilayer. The functional implication is of extensive promiscuity of initial ligand binding, primarily determined by size and shape of the ligand, with subsequent selection by unique interactions between atom groups of the ligand and the GPCR within the binding groove. This second step ensures the wide range of different efficacies documented for GPCR-targeting drugs. The NK1R data also provide a rationale for the observation that diffracting GPCR crystals are obtained for complexes with only very few of the ligands from libraries of approved drugs and lead compounds that bind to the receptors.

Neurokinins (or tachykinins) are peptides that modulate a wide variety of human physiology through the neurokinin G protein-coupled receptor family, implicated in a diverse array of pathological processes. Here we report high-resolution crystal structures of the human NK 1 receptor (NK 1 R) bound to two small-molecule antagonist therapeutics – aprepitant and netupitant and the progenitor antagonist CP-99,994. The structures reveal the detailed interactions between clinically approved antagonists and NK 1 R, which induce a distinct receptor conformation resulting in an interhelical hydrogen-bond network that cross-links the extracellular ends of helices V and VI. Furthermore, the high-resolution details of NK 1 R bound to netupitant establish a structural rationale for the lack of basal activity in NK 1 R. Taken together, these co-structures provide a comprehensive structural basis of NK 1 R antagonism and will facilitate the design of new therapeutics targeting the neurokinin receptor family. Neurokinin receptors are G protein-coupled receptors. Here the authors present three crystal structures of the neurokinin 1 receptor (NK 1 R) in complex with small-molecule antagonists including aprepitant and netupitant and observe that these clinically approved compounds induce a conformational change in the receptor.