ABSTRACT
The serotonin transporter (SERT) removes synaptic serotonin and is the target of anti-depressant drugs. SERT adopts three conformations: outward-open, occluded, and inward-open. All known inhibitors target the outward-open state except ibogaine, which has unusual anti-depressant and substance-withdrawal effects, and stabilizes the inward-open conformation. Unfortunately, ibogaine's promiscuity and cardiotoxicity limit the understanding of inward-open state ligands. We docked over 200 million small molecules against the inward-open state of the SERT. Thirty-six top-ranking compounds were synthesized, and thirteen inhibited; further structure-based optimization led to the selection of two potent (low nanomolar) inhibitors. These stabilized an outward-closed state of the SERT with little activity against common off-targets. A cryo-EM structure of one of these bound to the SERT confirmed the predicted geometry. In mouse behavioral assays, both compounds had anxiolytic- and anti-depressant-like activity, with potencies up to 200-fold better than fluoxetine (Prozac), and one substantially reversed morphine withdrawal effects.
Subject(s)
Ibogaine , Selective Serotonin Reuptake Inhibitors , Serotonin Plasma Membrane Transport Proteins , Small Molecule Libraries , Animals , Mice , Fluoxetine/pharmacology , Ibogaine/chemistry , Ibogaine/pharmacology , Molecular Conformation , Serotonin/metabolism , Serotonin Plasma Membrane Transport Proteins/chemistry , Serotonin Plasma Membrane Transport Proteins/metabolism , Serotonin Plasma Membrane Transport Proteins/ultrastructure , Selective Serotonin Reuptake Inhibitors/pharmacology , Small Molecule Libraries/pharmacologyABSTRACT
The σ2 receptor has attracted intense interest in cancer imaging1, psychiatric disease2, neuropathic pain3-5 and other areas of biology6,7. Here we determined the crystal structure of this receptor in complex with the clinical candidate roluperidone2 and the tool compound PB288. These structures templated a large-scale docking screen of 490 million virtual molecules, of which 484 compounds were synthesized and tested. We identified 127 new chemotypes with affinities superior to 1 µM, 31 of which had affinities superior to 50 nM. The hit rate fell smoothly and monotonically with docking score. We optimized three hits for potency and selectivity, and achieved affinities that ranged from 3 to 48 nM, with up to 250-fold selectivity versus the σ1 receptor. Crystal structures of two ligands bound to the σ2 receptor confirmed the docked poses. To investigate the contribution of the σ2 receptor in pain, two potent σ2-selective ligands and one potent σ1/σ2 non-selective ligand were tested for efficacy in a mouse model of neuropathic pain. All three ligands showed time-dependent decreases in mechanical hypersensitivity in the spared nerve injury model9, suggesting that the σ2 receptor has a role in nociception. This study illustrates the opportunities for rapid discovery of in vivo probes through structure-based screens of ultra large libraries, enabling study of underexplored areas of biology.
Subject(s)
Neuralgia , Receptors, sigma , Animals , Ligands , Mice , Neuralgia/drug therapy , Receptors, sigma/metabolism , Structure-Activity RelationshipABSTRACT
A remarkable molecular and functional heterogeneity of the primary sensory neurons and dorsal horn interneurons transmits pain- and or itch-relevant information, but the molecular signature of the projection neurons that convey the messages to the brain is unclear. Here, using retro-TRAP (translating ribosome affinity purification) and RNA sequencing, we reveal extensive molecular diversity of spino- and trigeminoparabrachial projection neurons. Among the many genes identified, we highlight distinct subsets of Cck+ -, Nptx2+ -, Nmb+ -, and Crh+ -expressing projection neurons. By combining in situ hybridization of retrogradely labeled neurons with Fos-based assays, we also demonstrate significant functional heterogeneity, including both convergence and segregation of pain- and itch-provoking inputs into molecularly diverse subsets of NK1R- and non-NK1R-expressing projection neurons.
Subject(s)
Neurons/pathology , Pain/complications , Pain/pathology , Pruritus/complications , Pruritus/pathology , Spinal Cord/pathology , Trigeminal Nerve/pathology , Animals , Chloroquine/pharmacology , Female , Gene Expression Regulation/drug effects , Male , Mice, Inbred C57BL , Neurons/drug effects , Neurons/metabolism , Pain/genetics , Physical Stimulation , Pruritus/genetics , RNA/isolation & purification , RNA/metabolism , Receptors, Neurokinin-1/metabolism , Spinal Cord Dorsal Horn/drug effects , Spinal Cord Dorsal Horn/metabolismABSTRACT
Pruritus is a common symptom of inflammatory skin conditions, including atopic dermatitis (AD). Although primary sensory neurons that transmit pruritic signals are well-cataloged, little is known about the neuronal alterations that occur as a result of skin disruption in AD. To address this question, we examined the molecular and behavioral consequences of challenging Grhl3PAR2/+ mice, which overexpress PAR2 in suprabasal keratinocytes, with serial topical application of the environmental allergen house dust mite (HDM). We monitored behavior and used RNA sequencing, qPCR, and in situ hybridization to evaluate gene expression in trigeminal ganglia (TG), before and after HDM. We found that neither Grhl3PAR2/+ nor wild-type (WT) mice exhibited spontaneous scratching, and pruritogen-induced acute scratching did not differ. In contrast, HDM exacerbated scratching in Grhl3PAR2/+ mice. Despite the absence of scratching in untreated Grhl3PAR2/+ mice, several TG genes in these mice were up-regulated compared to WT. HDM treatment of the Grhl3PAR2/+ mice enhanced up-regulation of this set of genes and induced additional genes, many within the subset of TG neurons that express TRPV1. The same set of genes was up-regulated in HDM-treated Grhl3PAR2/+ mice that did not scratch, but at lesser magnitude. Finally, we recorded comparable transcriptional changes in IL31Tg mice, demonstrating that a common genetic program is induced in two AD models. Taken together, we conclude that transcriptional changes that occur in primary sensory neurons in dermatitis-susceptible animals underlie a genetic priming that not only sensitizes the animal to chronic allergens but also contributes to pruritus in atopic skin disease.
Subject(s)
Allergens/toxicity , DNA-Binding Proteins/physiology , Dermatitis, Atopic/pathology , Receptor, PAR-2/metabolism , Sensory Receptor Cells/pathology , Skin/pathology , Transcription Factors/physiology , Animals , Dermatitis, Atopic/chemically induced , Dermatitis, Atopic/metabolism , Disease Models, Animal , Mice , Mice, Transgenic , RNA-Seq , Receptor, PAR-2/genetics , Sensory Receptor Cells/drug effects , Sensory Receptor Cells/metabolism , Skin/drug effects , Skin/innervation , Skin/metabolismABSTRACT
Febrile seizures (FSs) are the most common convulsion in infancy and childhood. Considering the limitations of current treatments, it is important to examine the mechanistic cause of FSs. Prompted by a genome-wide association study identifying TMEM16C (also known as ANO3) as a risk factor of FSs, we showed previously that loss of TMEM16C function causes hippocampal neuronal hyperexcitability [Feenstra et al., Nat. Genet. 46, 1274-1282 (2014)]. Our previous study further revealed a reduction in the number of warm-sensitive neurons that increase their action potential firing rate with rising temperature of the brain region harboring these hypothalamic neurons. Whereas central neuronal hyperexcitability has been implicated in FSs, it is unclear whether the maximal temperature reached during fever or the rate of body temperature rise affects FSs. Here we report that mutant rodent pups with TMEM16C eliminated from all or a subset of their central neurons serve as FS models with deficient thermoregulation. Tmem16c knockout (KO) rat pups at postnatal day 10 (P10) are more susceptible to hyperthermia-induced seizures. Moreover, they display a more rapid rise of body temperature upon heat exposure. In addition, conditional knockout (cKO) mouse pups (P11) with TMEM16C deletion from the brain display greater susceptibility of hyperthermia-induced seizures as well as deficiency in thermoregulation. We also found similar phenotypes in P11 cKO mouse pups with TMEM16C deletion from Ptgds-expressing cells, including temperature-sensitive neurons in the preoptic area (POA) of the anterior hypothalamus, the brain region that controls body temperature. These findings suggest that homeostatic thermoregulation plays an important role in FSs.
Subject(s)
Body Temperature Regulation/genetics , Chloride Channels/genetics , Fever/genetics , Hyperthermia/genetics , Preoptic Area/metabolism , Seizures, Febrile/genetics , Action Potentials/physiology , Animals , Animals, Newborn , Body Temperature/drug effects , Body Temperature/physiology , Chloride Channels/deficiency , Female , Fever/chemically induced , Fever/metabolism , Fever/physiopathology , Gene Expression , Hippocampus/metabolism , Hippocampus/physiopathology , Hyperthermia/metabolism , Hyperthermia/physiopathology , Kainic Acid/administration & dosage , Male , Mice , Mice, Knockout , Neurons/metabolism , Neurons/pathology , Preoptic Area/physiopathology , Protein Isoforms/deficiency , Protein Isoforms/genetics , Rats , Seizures, Febrile/chemically induced , Seizures, Febrile/metabolism , Seizures, Febrile/physiopathologyABSTRACT
A comprehensive study on the electronic spectral, photophysical and acid-base properties of phenyl- and methyl-oxime corrole derivatives and of triphenylcorrole (model corrole) has been performed, aiming to shed light on the existing species in the ground and excited states. Solvents and corrole concentration are found to govern the properties of the studied compounds and are determinants of their applicability in in vivo studies. In THF, the neutral corrole has two tautomeric forms (T1 and T2). In DMSO, the deprotonated form shows a characteristic long-wavelength Q band slightly shifted to blue when compared with the T1 tautomer and a higher fluorescence quantum yield. In ACN, with the increase of the corrole concentration formation of an aggregate due to homoconjugation (with dimer characteristics) is observed, and pioneeringly reported using UV-Vis and fluorescence studies and confirmed by carrying out titrations with TFA. The effect of the oxime group on the pK values of a corrole is found to influence the formation of a homoconjugate, namely by precluding its formation (at higher concentrations) when compared with the model corrole. TDDFT electronic quantum calculations support the experimental observations, namely the existence of tautomers and deprotonated species, with their respective electronic spectral features, further allowed proposing a structure for the homoconjugate complex in ACN. The characteristics of the oxime-corroles, namely a pK of â¼ 5, absorption and emission at ca. 650 nm and solvent dependent properties, make them good candidates for their use in biological systems either as probes, sensors, or as new sensitizers for photodynamic therapy.
ABSTRACT
Dysfunction of inhibitory circuits in the rostral anterior cingulate cortex underlies the affective (aversive), but not the sensory-discriminative features (hypersensitivity) of the pain experience. To restore inhibitory controls, we transplanted inhibitory interneuron progenitor cells into the rostral anterior cingulate cortex in a chemotherapy-induced neuropathic pain model. The transplants integrated, exerted a GABA-A mediated inhibition of host pyramidal cells and blocked gabapentin preference (i.e. relieved ongoing pain) in a conditioned place preference paradigm. Surprisingly, pain aversiveness persisted when the transplants populated both the rostral and posterior anterior cingulate cortex. We conclude that selective and long lasting inhibition of the rostral anterior cingulate cortex, in the mouse, has a profound pain relieving effect against nerve injury-induced neuropathic pain. However, the interplay between the rostral and posterior anterior cingulate cortices must be considered when examining circuits that influence ongoing pain and pain aversiveness.
Subject(s)
GABAergic Neurons/metabolism , GABAergic Neurons/transplantation , Gyrus Cinguli/metabolism , Neuralgia/metabolism , Neuralgia/therapy , Sciatic Neuropathy/therapy , Animals , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neuralgia/pathology , Receptors, GABA-B/metabolism , Sciatic Neuropathy/metabolism , Sciatic Neuropathy/pathologyABSTRACT
BACKGROUND: Despite recent insights into the pathophysiology of acute and chronic itch, chronic itch remains an often intractable condition. Among major contributors to chronic itch is dysfunction of spinal cord gamma aminobutyric acidergic (GABAergic) inhibitory controls. OBJECTIVES: We sought to test the hypothesis that selective GABA agonists as well as cell transplant-derived GABA are antipruritic against acute itch and in a transgenic mouse model of atopic dermatitis produced by overexpression of the TH2 cell-associated cytokine, IL-31 (IL-31Tg mice). METHODS: We injected wild-type and IL-31Tg mice with combinations of GABA-A (muscimol) or GABA-B (baclofen) receptor agonists 15 to 20 minutes prior to injection of various pruritogens (histamine, chloroquine, or endothelin-1) and recorded spontaneous scratching before and after drug administration. We also tested the antipruritic properties of intraspinal transplantation of precursors of GABAergic interneurons in the IL-31Tg mice. RESULTS: Systemic muscimol or baclofen are antipruritic against both histamine-dependent and -independent pruritogens, but the therapeutic window using either ligand alone was very small. In contrast, combined subthreshold doses of baclofen and muscimol produced a significant synergistic antipruritic effect, with no sedation. Finally, transplant-mediated long-term enhancement of GABAergic signaling not only reduced spontaneous scratching in the IL-31Tg mice but also dramatically resolved the associated skin lesions. CONCLUSIONS: Although additional research is clearly needed, existing approved GABA agonists should be considered in the management of chronic itch, notably atopic dermatitis.
Subject(s)
Antipruritics/therapeutic use , Baclofen/therapeutic use , Dermatitis, Atopic/drug therapy , GABA-A Receptor Agonists/therapeutic use , GABA-B Receptor Agonists/therapeutic use , Muscimol/therapeutic use , Animals , Dermatitis, Atopic/metabolism , Dermatitis, Atopic/therapy , Disease Models, Animal , Drug Synergism , Gastrin-Releasing Peptide/genetics , Glutamate Decarboxylase/genetics , Interleukins/genetics , Interneurons/drug effects , Male , Median Eminence/cytology , Mice, Inbred C57BL , Mice, Transgenic , RNA, Messenger/metabolism , Receptors, Bombesin/genetics , Receptors, GABA-A/genetics , Receptors, GABA-B/genetics , Receptors, Neurokinin-1/genetics , Skin/drug effects , Skin/pathology , Spinal Cord/drug effects , Spinal Cord/metabolism , Stem Cell TransplantationABSTRACT
Spinal cord transplants of embryonic cortical GABAergic progenitor cells derived from the medial ganglionic eminence (MGE) can reverse mechanical hypersensitivity in the mouse models of peripheral nerve injury- and paclitaxel-induced neuropathic pain. Here, we used electrophysiology, immunohistochemistry, and electron microscopy to examine the extent to which MGE cells integrate into host circuitry and recapitulate endogenous inhibitory circuits. Whether the transplants were performed before or after nerve injury, the MGE cells developed into mature neurons and exhibited firing patterns characteristic of subpopulations of cortical and spinal cord inhibitory interneurons. Conversely, the transplanted cells preserved cortical morphological and neurochemical properties. We also observed a robust anatomical and functional synaptic integration of the transplanted cells into host circuitry in both injured and uninjured animals. The MGE cells were activated by primary afferents, including TRPV1-expressing nociceptors, and formed GABAergic, bicuculline-sensitive, synapses onto host neurons. Unexpectedly, MGE cells transplanted before injury prevented the development of mechanical hypersensitivity. Together, our findings provide direct confirmation of an extensive, functional synaptic integration of MGE cells into host spinal cord circuits. This integration underlies normalization of the dorsal horn inhibitory tone after injury and may be responsible for the prophylactic effect of preinjury transplants. SIGNIFICANCE STATEMENT: Spinal cord transplants of embryonic cortical GABAergic interneuron progenitors from the medial ganglionic eminence (MGE), can overcome the mechanical hypersensitivity produced in different neuropathic pain models in adult mice. Here, we examined the properties of transplanted MGE cells and the extent to which they integrate into spinal cord circuitry. Using electrophysiology, immunohistochemistry, and electron microscopy, we demonstrate that MGE cells, whether transplanted before or after nerve injury, develop into inhibitory neurons, are activated by nociceptive primary afferents, and form GABA-A-mediated inhibitory synapses with the host. Unexpectedly, cells transplanted into naive spinal cord prevented the development of nerve-injury-induced mechanical hypersensitivity. These results illustrate the remarkable plasticity of adult spinal cord and the potential of cell-based therapies against neuropathic pain.
Subject(s)
GABAergic Neurons/pathology , Hyperalgesia/physiopathology , Hyperalgesia/therapy , Neural Stem Cells/transplantation , Spinal Cord Regeneration/physiology , Spinal Cord/physiology , Synapses/pathology , Animals , GABAergic Neurons/metabolism , Hyperalgesia/pathology , Male , Mice , Mice, Inbred C57BL , Prosencephalon/cytology , Stem Cell Transplantation/methods , Synapses/metabolism , Treatment OutcomeABSTRACT
There is continuing controversy relating to the primary afferent neurotransmitter that conveys itch signals to the spinal cord. Here, we investigated the DRG and spinal cord expression of the putative primary afferent-derived "itch" neurotransmitter, gastrin-releasing peptide (GRP). Using ISH, qPCR, and immunohistochemistry, we conclude that GRP is expressed abundantly in spinal cord, but not in DRG neurons. Titration of the most commonly used GRP antiserum in tissues from wild-type and GRP mutant mice indicates that the antiserum is only selective for GRP at high dilutions. Paralleling these observations, we found that a GRPeGFP transgenic reporter mouse has abundant expression in superficial dorsal horn neurons, but not in the DRG. In contrast to previous studies, neither dorsal rhizotomy nor an intrathecal injection of capsaicin, which completely eliminated spinal cord TRPV1-immunoreactive terminals, altered dorsal horn GRP immunoreactivity. Unexpectedly, however, peripheral nerve injury induced significant GRP expression in a heterogeneous population of DRG neurons. Finally, dual labeling and retrograde tracing studies showed that GRP-expressing neurons of the superficial dorsal horn are predominantly interneurons, that a small number coexpress protein kinase C gamma (PKCγ), but that none coexpress the GRP receptor (GRPR). Our studies support the view that pruritogens engage spinal cord "itch" circuits via excitatory superficial dorsal horn interneurons that express GRP and that likely target GRPR-expressing interneurons. The fact that peripheral nerve injury induced de novo GRP expression in DRG neurons points to a novel contribution of this peptide to pruritoceptive processing in neuropathic itch conditions.
Subject(s)
Gastrin-Releasing Peptide/metabolism , Neurons, Afferent/metabolism , Spinal Cord/metabolism , Animals , Antibodies/immunology , Ganglia, Spinal/cytology , Ganglia, Spinal/metabolism , Gastrin-Releasing Peptide/genetics , Gastrin-Releasing Peptide/immunology , Immunochemistry/methods , Immunochemistry/standards , Male , Mice , Mice, Inbred C57BL , Organ Specificity , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sensitivity and Specificity , TRPV Cation Channels/genetics , TRPV Cation Channels/metabolismABSTRACT
Most reports agree that aging negatively impacts pain processing and that the prevalence of chronic pain increases significantly with age. To improve current therapies, it is critical that aged animals be included in preclinical studies. Here we compared sensitivities to pain and itch-provoking stimuli in naïve and injured young and aged mice. Surprisingly, we found that in the absence of injury, aged male and female mice are significantly less responsive to mechanical stimuli and, in females, also to noxious thermal (heat) stimuli. In both older male and female mice, compared to younger (6-month-old mice), we also recorded reduced pruritogen-evoked scratching. On the other hand, after nerve injury, aged mice nevertheless developed significant mechanical hypersensitivity. Interestingly, however, and in contrast to young mice, aged mice developed both ipsilateral and contralateral postinjury mechanical allodynia. In a parallel immunohistochemical analysis of microglial and astrocyte markers, we found that the ipsilateral to the contralateral ratio of nerve injury-induced expression decreased with age. That observation is consistent with our finding of contralateral hypersensitivity after nerve injury in the aged but not the young mice. We conclude that aging has opposite effects on baseline versus postinjury pain and itch processing. PERSPECTIVE: Aged male and female mice (22-24 months) are less sensitive to mechanical, thermal (heat), and itch-provoking stimuli than are younger mice (6 months).
Subject(s)
Pain , Pruritus , Male , Female , Mice , Animals , Hyperalgesia/etiologyABSTRACT
T cells have emerged as sex-dependent orchestrators of pain chronification but the sexually dimorphic mechanisms by which T cells control pain sensitivity is not resolved. Here, we demonstrate an influence of regulatory T cells (Tregs) on pain processing that is distinct from their canonical functions of immune regulation and tissue repair. Specifically, meningeal Tregs (mTregs) express the endogenous opioid, enkephalin, and mTreg-derived enkephalin exerts an antinociceptive action through a presynaptic opioid receptor signaling mechanism that is dispensable for immunosuppression. mTregs are both necessary and sufficient for suppressing mechanical pain sensitivity in female but not male mice. Notably, the mTreg modulation of pain thresholds depends on sex-hormones and expansion of enkephalinergic mTregs during gestation imparts a remarkable pregnancy-induced analgesia in a pre-existing, chronic, unremitting neuropathic pain model. These results uncover a fundamental sex-specific, pregnancy-pronounced, and immunologically-derived endogenous opioid circuit for nociceptive regulation with critical implications for pain biology and maternal health.
ABSTRACT
Large library docking can reveal unexpected chemotypes that complement the structures of biological targets. Seeking new agonists for the cannabinoid-1 receptor (CB1R), we docked 74 million tangible molecules, prioritizing 46 high ranking ones for de novo synthesis and testing. Nine were active by radioligand competition, a 20% hit-rate. Structure-based optimization of one of the most potent of these (Ki = 0.7 uM) led to '4042, a 1.9 nM ligand and a full CB1R agonist. A cryo-EM structure of the purified enantiomer of '4042 ('1350) in complex with CB1R-Gi1 confirmed its docked pose. The new agonist was strongly analgesic, with generally a 5-10-fold therapeutic window over sedation and catalepsy and no observable conditioned place preference. These findings suggest that new cannabinoid chemotypes may disentangle characteristic cannabinoid side-effects from their analgesia, supporting the further development of cannabinoids as pain therapeutics.
ABSTRACT
The general consensus is that increases in neuronal activity in the anterior cingulate cortex (ACC) contribute to pain's negative affect. Here, using in vivo imaging of neuronal calcium dynamics in mice, we report that nitrous oxide, a general anesthetic that reduces pain affect, paradoxically, increases ACC spontaneous activity. As expected, a noxious stimulus also increased ACC activity. However, as nitrous oxide increases baseline activity, the relative change in activity from pre-stimulus baseline was significantly less than the change in the absence of the general anesthetic. We suggest that this relative change in activity represents a neural signature of the affective pain experience. Furthermore, this signature of pain persists under general anesthesia induced by isoflurane, at concentrations in which the mouse is unresponsive. We suggest that this signature underlies the phenomenon of connected consciousness, in which use of the isolated forelimb technique revealed that pain percepts can persist in anesthetized patients.
ABSTRACT
ABSTRACT: Altered bone morphogenetic protein (BMP) signaling is associated with many musculoskeletal diseases. However, it remains unknown whether BMP dysfunction has direct contribution to debilitating pain reported in many of these disorders. Here, we identified a novel neuropathic pain phenotype in patients with fibrodysplasia ossificans progressiva (FOP), a rare autosomal-dominant musculoskeletal disorder characterized by progressive heterotopic ossification. Ninety-seven percent of these patients carry an R206H gain-of-function point mutation in the BMP type I receptor ACVR1 (ACVR1 R206H ), which causes neofunction to Activin A and constitutively activates signaling through phosphorylated SMAD1/5/8. Although patients with FOP can harbor pathological lesions in the peripheral and central nervous system, their etiology and clinical impact are unclear. Quantitative sensory testing of patients with FOP revealed significant heat and mechanical pain hypersensitivity. Although there was no major effect of ACVR1 R206H on differentiation and maturation of nociceptive sensory neurons (iSNs) derived from FOP induced pluripotent stem cells, both intracellular and extracellular electrophysiology analyses of the ACVR1 R206H iSNs displayed ACVR1-dependent hyperexcitability, a hallmark of neuropathic pain. Consistent with this phenotype, we recorded enhanced responses of ACVR1 R206H iSNs to TRPV1 and TRPA1 agonists. Thus, activated ACVR1 signaling can modulate pain processing in humans and may represent a potential target for pain management in FOP and related BMP pathway diseases.
Subject(s)
Myositis Ossificans , Neuralgia , Ossification, Heterotopic , Humans , Gain of Function Mutation , Ossification, Heterotopic/genetics , Ossification, Heterotopic/metabolism , Ossification, Heterotopic/pathology , Myositis Ossificans/genetics , Myositis Ossificans/metabolism , Myositis Ossificans/pathology , Sensory Receptor Cells/metabolism , Neuralgia/genetics , Mutation/genetics , Activin Receptors, Type I/genetics , Activin Receptors, Type I/metabolismABSTRACT
Despite robust literature associating IL-31 with pruritic inflammatory skin diseases, its influence on cutaneous inflammation and the interplay between inflammatory and neurosensory pathways remain unmapped. Here, we examined the consequences of disrupting Il31 and its receptor Il31ra in a mouse model of house dust mite (HDM)-induced allergic dermatitis. Il31-deficient mice displayed a deficit in HDM dermatitis-associated scratching, consistent with its well-established role as a pruritogen. In contrast, Il31 deficiency increased the number and proportion of cutaneous type 2 cytokine-producing CD4+ T cells and serum IgE in response to HDM. Furthermore, Il4ra+ monocytes and macrophages capable of fueling a feedforward type 2 inflammatory loop were selectively enriched in Il31ra-deficient HDM dermatitis skin. Thus, IL-31 is not strictly a proinflammatory cytokine but rather an immunoregulatory factor that limits the magnitude of type 2 inflammatory responses in skin. Our data support a model wherein IL-31 activation of IL31RA+ pruritoceptors triggers release of calcitonin gene-related protein (CGRP), which can mediate neurogenic inflammation, inhibit CD4+ T cell proliferation, and reduce T cell production of the type 2 cytokine IL-13. Together, these results illustrate a previously unrecognized neuroimmune pathway that constrains type 2 tissue inflammation in the setting of chronic cutaneous allergen exposure and may explain paradoxical dermatitis flares in atopic patients treated with anti-IL31RA therapy.
Subject(s)
Dermatitis, Atopic , Neurogenic Inflammation , Animals , Mice , Cytokines , Immunity , Pyroglyphidae , Skin/immunology , Interleukins/immunology , Interleukins/metabolismABSTRACT
The lipid prostaglandin E2 (PGE2) mediates inflammatory pain by activating G protein-coupled receptors, including the prostaglandin E2 receptor 4 (EP4R). Nonsteroidal anti-inflammatory drugs (NSAIDs) reduce nociception by inhibiting prostaglandin synthesis, however, the disruption of upstream prostanoid biosynthesis can lead to pleiotropic effects including gastrointestinal bleeding and cardiac complications. In contrast, by acting downstream, EP4R antagonists may act specifically as anti-inflammatory agents and, to date, no selective EP4R antagonists have been approved for human use. In this work, seeking to diversify EP4R antagonist scaffolds, we computationally dock over 400 million compounds against an EP4R crystal structure and experimentally validate 71 highly ranked, de novo synthesized molecules. Further, we show how structure-based optimization of initial docking hits identifies a potent and selective antagonist with 16 nanomolar potency. Finally, we demonstrate favorable pharmacokinetics for the discovered compound as well as anti-allodynic and anti-inflammatory activity in several preclinical pain models in mice.
Subject(s)
Dinoprostone , Receptors, Prostaglandin , Humans , Mice , Animals , Phagocytosis , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Pain/drug therapy , Anti-Inflammatory Agents, Non-Steroidal/pharmacologyABSTRACT
Primary afferent "pain" fibers (nociceptors) are divided into subclasses based on distinct molecular and anatomical features, and these classes mediate noxious modality-specific contributions to behaviors evoked by painful stimuli. Whether the heat and capsaicin receptor transient receptor potential vanilloid-1 (TRPV1) is expressed heterogeneously across several sensory populations, or is selectively expressed by a unique nociceptor subclass, however, is unclear. Here we used two lines of Trpv1 reporter mice to investigate the primary afferent expression of TRPV1, both during development and in the adult. We demonstrate, using Cre-induced lineage tracing, that during development TRPV1 is transiently expressed in a wide range of dorsal root ganglion neurons, and that its expression is gradually refined, such that TRPV1 transcripts become restricted to a specific subset of peptidergic sensory neurons. Finally, the remarkable sensitivity that is characteristic of these reporter mice revealed an innervation of central and peripheral targets by TRPV1+ primary afferents in the adult that is considerably more extensive than has previously been appreciated.
Subject(s)
Ganglia, Spinal/metabolism , Neurons, Afferent/metabolism , Neuropeptides/metabolism , TRPV Cation Channels/metabolism , Animals , Gene Expression Regulation, Developmental , Mice , Mice, Transgenic , Nociceptors/metabolism , TRPV Cation Channels/geneticsABSTRACT
TRPV1, a capsaicin- and heat-activated ion channel, is expressed by peripheral nociceptors and has been implicated in various inflammatory and neuropathic pain conditions. Although pharmacological modulation of TRPV1 has attracted therapeutic interest, many TRPV1 agonists and antagonists produce thermomodulatory side effects in animal models and human clinical trials, limiting their utility. These on-target effects may result from the perturbation of TRPV1 receptors on nociceptors, which transduce signals to central thermoregulatory circuits and release proinflammatory factors from their peripheral terminals, most notably the potent vasodilative neuropeptide, calcitonin gene-related peptide (CGRP). Alternatively, these body temperature effects may originate from the modulation of TRPV1 on vascular smooth muscle cells (vSMCs), where channel activation promotes arteriole constriction. Here, we ask which of these pathways is most responsible for the body temperature perturbations elicited by TRPV1 drugs in vivo. We address this question by selectively eliminating TRPV1 expression in sensory neurons or vSMCs and show that only the former abrogates agonist-induced hypothermia and antagonist-induced hyperthermia. Furthermore, lesioning the central projections of TRPV1-positive sensory nerve fibers also abrogates drug-mediated thermomodulation, whereas eliminating CGRP has no effect. Thus, TRPV1 drugs alter core body temperature by modulating sensory input to the central nervous system, rather than through peripheral actions on the vasculature. These findings suggest how mechanistically distinct TRPV1 antagonists may diminish inflammatory pain without affecting core body temperature.
Subject(s)
Body Temperature , Neuralgia , Animals , Calcitonin Gene-Related Peptide , Capsaicin/pharmacology , Humans , Sensory Receptor Cells , TRPV Cation ChannelsABSTRACT
The 5-HT5A receptor (5-HT5AR), for which no selective agonists and a few antagonists exist, remains the least understood serotonin receptor. A single commercial antagonist, SB-699551, has been widely used to investigate the 5-HT5AR function in neurological disorders, including pain, but this molecule has substantial liabilities as a chemical probe. Accordingly, we sought to develop an internally controlled probe set. Docking over 6 million molecules against a 5-HT5AR homology model identified 5 mid-µM ligands, one of which was optimized to UCSF678, a 42 nM arrestin-biased partial agonist at the 5-HT5AR with a more restricted off-target profile and decreased assay liabilities versus SB-699551. Site-directed mutagenesis supported the docked pose of UCSF678. Surprisingly, analogs of UCSF678 that lost the 5-HT5AR activity revealed that 5-HT5AR engagement is nonessential for alleviating pain, contrary to studies with less-selective ligands. UCSF678 and analogs constitute a selective probe set with which to study the function of the 5-HT5AR.