Inhibiting spinal cord-specific hsp90 isoforms reveals a novel strategy to improve the therapeutic index of opioid treatment.

Opioids are the gold standard for the treatment of chronic pain but are limited by adverse side effects. In our earlier work, we showed that Heat shock protein 90 (Hsp90) has a crucial role in regulating opioid signaling in spinal cord; Hsp90 inhibition in spinal cord enhances opioid anti-nociception. Building on these findings, we injected the non-selective Hsp90 inhibitor KU-32 by the intrathecal route into male and female CD-1 mice, showing that morphine anti-nociceptive potency was boosted by 1.9-3.5-fold in acute and chronic pain models. At the same time, tolerance was reduced from 21-fold to 2.9 fold and established tolerance was rescued, while the potency of constipation and reward was unchanged. These results demonstrate that spinal Hsp90 inhibition can improve the therapeutic index of morphine. However, we also found that systemic non-selective Hsp90 inhibition blocked opioid pain relief. To avoid this effect, we used selective small molecule inhibitors and CRISPR gene editing to identify 3 Hsp90 isoforms active in spinal cord (Hsp90α, Hsp90ß, and Grp94) while only Hsp90α was active in brain. We thus hypothesized that a systemically delivered selective inhibitor to Hsp90ß or Grp94 could selectively inhibit spinal cord Hsp90 activity, resulting in enhanced opioid therapy. We tested this hypothesis using intravenous delivery of KUNB106 (Hsp90ß) and KUNG65 (Grp94), showing that both drugs enhanced morphine anti-nociceptive potency while rescuing tolerance. Together, these results suggest that selective inhibition of spinal cord Hsp90 isoforms is a novel, translationally feasible strategy to improve the therapeutic index of opioids.

Select Minor Cannabinoids from Cannabis sativa are Cannabimimetic and Antinociceptive in a Mouse Model of Chronic Neuropathic Pain.

Chronic pain conditions affect nearly 20% of the population in the United States. Current medical interventions, such as opioid drugs, are effective at relieving pain but are accompanied by many undesirable side effects. This is one reason increased numbers of chronic pain patients have been turning to Cannabis for pain management. Cannabis contains many bioactive chemical compounds; however, current research looking into lesser-studied minor cannabinoids in Cannabis lacks uniformity between experimental groups and/or excludes female mice from investigation. This makes it challenging to draw conclusions between experiments done with different minor cannabinoid compounds between labs or parse out potential sex differences that could be present. We chose five minor cannabinoids found in lower quantities within Cannabis: cannabinol (CBN), cannabidivarin (CBDV), cannabigerol (CBG), Δ8-tetrahydrocannabinol (Δ8-THC), and Δ9-tetrahydrocannabivarin (THCV). These compounds were then tested for their cannabimimetic and pain-relieving behaviors in a cannabinoid tetrad assay and a chemotherapy-induced peripheral neuropathy (CIPN) pain model in male and female CD-1 mice. We found that the minor cannabinoids we tested differed in the cannabimimetic behaviors evoked, as well as the extent. We found that CBN, CBG, and high dose Δ8-THC evoked some tetrad behaviors in both sexes, while THCV and low dose Δ8-THC exhibited cannabimimetic tetrad behaviors only in females. Only CBN efficaciously relieved CIPN pain, which contrasts with reports from other researchers. Together these findings provide further clarity to the pharmacology of minor cannabinoids and suggest further investigation into their mechanism and therapeutic potential. Significance Statement Minor cannabinoids are poorly studied ligands present in lower levels in Cannabis than cannabinoids like THC. In this study we evaluated 5 minor cannabinoids (CBN, CBDV, CBG, THCV, and Δ8-THC) for their cannabimimetic and analgesic effects in mice. We found that 4 of the 5 minor cannabinoids showed cannabimimetic activity, while one was efficacious in relieving chronic neuropathic pain. This work is important in further evaluating the activity of these drugs, which are seeing wider public use with marijuana legalization.

Terpenes from Cannabis sativa induce antinociception in a mouse model of chronic neuropathic pain via activation of adenosine A2A receptors.

ABSTRACT: Terpenes are small hydrocarbon compounds that impart aroma and taste to many plants, including Cannabis sativa. A number of studies have shown that terpenes can produce pain relief in various pain states in both humans and animals. However, these studies were methodologically limited and few established mechanisms of action. In our previous work, we showed that the terpenes geraniol, linalool, ß-pinene, α-humulene, and ß-caryophyllene produced cannabimimetic behavioral effects via multiple receptor targets. We thus expanded this work to explore the potential antinociception and mechanism of these Cannabis terpenes in a mouse model of chronic pain. We first tested for antinociception by injecting terpenes (200 mg/kg, IP) into male and female CD-1 mice with mouse models of chemotherapy-induced peripheral neuropathy (CIPN) or lipopolysaccharide-induced inflammatory pain, finding that the terpenes produced roughly equal antinociception to 10 mg/kg morphine or 3.2 mg/kg WIN55,212. We further found that none of the terpenes produced reward as measured by conditioned place preference, while low doses of terpene (100 mg/kg) combined with morphine (3.2 mg/kg) produced enhanced antinociception vs either alone. We then used the adenosine A2A receptor (A2AR) selective antagonist istradefylline (3.2 mg/kg, IP) and spinal cord-specific CRISPR knockdown of the A2AR to identify this receptor as the mechanism for terpene antinociception in CIPN. In vitro cAMP and binding studies and in silico modeling studies further suggested that the terpenes act as A2AR agonists. Together these studies identify Cannabis terpenes as potential therapeutics for chronic neuropathic pain and identify a receptor mechanism for this activity.

The Role of Ryanodine Receptor 2 in Drug-Associated Learning.

Type-2 ryanodine receptor (RyR2) ion channels facilitate the release of Ca 2+ from stores and serve an important function in neuroplasticity. The role for RyR2 in hippocampal-dependent learning and memory is well established and chronic hyperphosphorylation of RyR2 (RyR2P) is associated with pathological calcium leakage and cognitive disorders, including Alzheimer's disease. By comparison, little is known about the role of RyR2 in the ventral medial prefrontal cortex (vmPFC) circuitry important for working memory, decision making, and reward seeking. Here, we evaluated the basal expression and localization of RyR2 and RyR2P in the vmPFC. Next, we employed an operant model of sucrose, cocaine, or morphine self-administration (SA) followed by a (reward-free) recall test, to reengage vmPFC neurons and reactivate reward-seeking and re-evaluated the expression and localization of RyR2 and RyR2P in vmPFC. Under basal conditions, RyR2 was expressed in pyramidal cells but not regularly detected in PV/SST interneurons. On the contrary, RyR2P was rarely observed in PFC somata and was restricted to a different subcompartment of the same neuron - the apical dendrites of layer-5 pyramidal cells. Chronic SA of drug (cocaine or morphine) and nondrug (sucrose) rewards produced comparable increases in RyR2 protein expression. However, recalling either drug reward impaired the usual localization of RyR2P in dendrites and markedly increased its expression in somata immunoreactive for Fos, a marker of highly activated neurons. These effects could not be explained by chronic stress or drug withdrawal and instead appeared to require a recall experience associated with prior drug SA. In addition to showing the differential distribution of RyR2/RyR2P and affirming the general role of vmPFC in reward learning, this study provides information on the propensity of addictive drugs to redistribute RyR2P ion channels in a neuronal population engaged in drug-seeking. Hence, focusing on the early impact of addictive drugs on RyR2 function may serve as a promising approach to finding a treatment for substance use disorders.

Increases in local skin temperature correlate with spontaneous foot lifting and heat hyperalgesia in both incisional inflammatory models of pain.

Background: This study investigated if a localized increase in skin temperature in rat models of incisional and inflammatory pain correlates with the intensity of spontaneous and evoked pain behaviors. Methods: Anesthetized rats received either a 20-mm longitudinal incision made through the skin, fascia, and muscle of the plantar hind paw or an injection of complete Freund adjuvant into the plantar hind paw of anesthetized rats to induce local inflammation. Spontaneous and evoked pain behaviors were assessed, and changes in skin temperature were measured using a noncontact infrared thermometer. Results: There were no differences in skin temperature between the ipsilateral and contralateral hind paw before the incision or inflammation. Skin temperature increased at 2 hours after hind paw plantar incision or 1 day after inflammation of the affected paw, which gradually returned to baseline by the first day and fourth days after treatment, respectively. The increase in skin temperature correlated with the intensity of spontaneous pain behaviors and heat but not with mechanical allodynia. Conclusions: Our results suggest that a simple measurement of localized skin temperature using a noncontact infrared thermometer could measure the extent of spontaneous pain behaviors and heat hyperalgesia following plantar incision or inflammation in animals. In the absence of a reliable objective marker of pain, these results are encouraging. However, studies are warranted to validate our results using analgesics and pain-relieving interventions, such as nerve block on skin temperature changes.

Terpenes from Cannabis sativa Induce Antinociception in Mouse Chronic Neuropathic Pain via Activation of Spinal Cord Adenosine A2A Receptors.

Terpenes are small hydrocarbon compounds that impart aroma and taste to many plants, including Cannabis sativa. A number of studies have shown that terpenes can produce pain relief in various pain states in both humans and animals. However, these studies were methodologically limited and few established mechanisms of action. In our previous work, we showed that the terpenes geraniol, linalool, ß-pinene, α-humulene, and ß-caryophyllene produced cannabimimetic behavioral effects via multiple receptor targets. We thus expanded this work to explore the efficacy and mechanism of these Cannabis terpenes in relieving chronic pain. We first tested for antinociceptive efficacy by injecting terpenes (200 mg/kg, IP) into male and female CD-1 mice with chemotherapy-induced peripheral neuropathy (CIPN) or lipopolysaccharide-induced inflammatory pain, finding that the terpenes produced roughly equal efficacy to 10 mg/kg morphine or 3.2 mg/kg WIN55,212. We further found that none of the terpenes produced reward as measured by conditioned place preference, while low doses of terpene (100 mg/kg) combined with morphine (3.2 mg/kg) produced enhanced antinociception vs. either alone. We then used the adenosine A2A receptor (A2AR) selective antagonist istradefylline (3.2 mg/kg, IP) and spinal cord-specific CRISPR knockdown of the A2AR to identify this receptor as the mechanism for terpene antinociception in CIPN. In vitro cAMP and binding studies and in silico modeling studies further suggested that the terpenes act as A2AR agonists. Together these studies identify Cannabis terpenes as potential therapeutics for chronic neuropathic pain, and identify a receptor mechanism in the spinal cord for this activity.

HSP90 inhibition in the mouse spinal cord enhances opioid signaling by suppressing an AMPK-mediated negative feedback loop.

Opioids and other agonists of the µ-opioid receptor are effective at managing acute pain, but their chronic use can lead to tolerance that limits their efficacy. We previously reported that inhibiting the chaperone protein HSP90 in the spinal cords of mice promotes the antinociceptive effects of opioids in a manner that involved increased activation of the kinase ERK. Here, we found that the underlying mechanism involves the relief of a negative feedback loop mediated by the kinase AMPK. Intrathecal treatment of male and female mice with the HSP90 inhibitor 17-AAG decreased the abundance of the ß1 subunit of AMPK in the spinal cord. The antinociceptive effects of 17-AAG with morphine were suppressed by intrathecal administration of AMPK activators and enhanced by an AMPK inhibitor. Opioid treatment increased the abundance of phosphorylated AMPK in the dorsal horn of the spinal cord, where it colocalized with a neuronal marker and the neuropeptide CGRP. Knocking down AMPK in CGRP-positive neurons enhanced the antinociceptive effects of morphine and demonstrated that AMPK mediated the signal transduction between HSP90 inhibition and ERK activation. These data suggest that AMPK mediates an opioid-induced negative feedback loop in CGRP neurons of the spinal cord and that this loop can be disabled by HSP90 inhibition to enhance the efficacy of opioids.

The D3 receptor antagonist SR 21502 reduces cue-induced reinstatement of methamphetamine-seeking in rats.

There is as of yet no FDA-approved medication for methamphetamine use disorder. Although dopamine D3 receptor antagonists have been shown to be useful in reducing methamphetamine seeking in animal models their translation to the clinic has been hindered because currently tested compounds can produce dangerously high blood pressure. Thus, it is important to continue to explore other classes of D3 antagonists. We report here the effects of SR 21502, a selective D3 receptor antagonist, on cue-induced reinstatement (i.e., relapse) of methamphetamine-seeking in rats. In Experiment 1, rats were trained to self-administer methamphetamine under a fixed ratio schedule of reinforcement followed by extinction of the response. Then, animals were tested with one of several doses of SR 21502 on cue-induced reinstatement of responding. SR 21502 significantly reduced cue-induced reinstatement of methamphetamine-seeking. In Experiment 2, animals were trained to lever press for food under a PR schedule and tested with the lowest dose of SR 21502 that caused a significant reduction in Experiment 1. These animals responded on average 8 times more than the vehicle-treated rats in Experiment 1, eliminating the possibility that SR 21502-treated rats in Experiment 1 responded less because they were incapacitated. In summary, these data suggest that SR 21502 may selectively inhibit methamphetamine-seeking and may constitute a promising pharmacotherapeutic agent for methamphetamine or other drug use disorders.

The role of opioid receptors in modulating Alzheimer's Disease.

Alzheimer's disease (AD) is a complex neurological disorder characterized by accumulation of amyloid plaques and neurofibrillary tangles. Long term investigation of AD pathogenesis suggests that ß-site amyloid precursor protein [APP] cleaving enzyme 1 (BACE1) and γ-secretase enzymes promote the amyloidogenic pathway and produce toxic Aß peptides that are predisposed to aggregate in the brain. Hence, the targeted inhibition of BACE1/γ-secretase expression and function is a promising approach for AD therapy. Several reports have suggested that the opioid family of G-protein coupled receptors modulate the etiology of AD progression. It has also been found that changes in the signaling pathways of opioid receptors increased the expression of BACE1 and γ-secretase, and is strongly correlated with abnormal production of Aß and pathogenesis of AD. Thus, the opioid receptor family is a promising candidate for targeted drug development to treat AD. In this review, we outline the involvement and mechanisms of opioid receptor signaling modulation in Alzheimer's Disease progression.

Structure-Based Design of Glycosylated Oxytocin Analogues with Improved Selectivity and Antinociceptive Activity.

Acute and chronic pain is often treated with opioids despite the negative side effects of constipation, physical dependence, respiratory depression, and overdose. The misuse of opioid analgesics has given rise to the opioid crisis/epidemic, and alternate nonaddictive analgesics are urgently needed. Oxytocin, a pituitary hormone, is an alternative to the small molecule treatments available and has been used as an analgesic as well as for the treatment and prevention of opioid use disorder (OUD). Clinical implementation is limited by its poor pharmacokinetic profile, a result of the labile disulfide bond between two cysteine residues in the native sequence. Stable brain penetrant oxytocin analogues have been synthesized by replacement of the disulfide bond with a stable lactam and glycosidation of the C-terminus. These analogues show exquisite selectivity for the oxytocin receptor and potent in vivo antinociception in mice following peripheral (i.v.) administration, supporting further study of their clinical potential.

Design of Analgesic Trivalent Peptides with Low Withdrawal Symptoms: Probing the Antinociceptive Profile of Novel Linear and Cyclic Peptides as Opioid Pan Ligands.

The discovery of efficacious and safe analgesics with reduced side effects is the foremost challenge in the pain field. In this work, we report the in vitro and in vivo evaluation of linear and cyclic analogues of biphalin with the aim to complete the series of structural modifications previously applied in the development of opioid peptides incorporating a xylene bridge. Replacement of Tyr1,1' by Dmt (2,5-dimethyltyrosine) in the linear biphalin analogue AM94 and cyclic analogue MACE4 resulted in two new compounds (namely, MJ2 and MJ5) endowed with improved KOR/MOR/DOR binding affinity. Both compounds showed a strong antinociceptive profile in in vivo models of nociception, allodynia, and hyperalgesia via the tail flick, hot plate, and formalin tests after intracerebroventricular and subcutaneous administration. One of these ligands, MJ2, was also tested in tolerance and dependence studies, exhibiting very little withdrawal symptoms.

Strategies towards safer opioid analgesics-A review of old and upcoming targets.

Opioids continue to be of use for the treatment of pain. Most clinically used analgesics target the µ opioid receptor whose activation results in adverse effects like respiratory depression, addiction and abuse liability. Various approaches have been used by the field to separate receptor-mediated analgesic actions from adverse effects. These include biased agonism, opioids targeting multiple receptors, allosteric modulators, heteromers and splice variants of the µ receptor. This review will focus on the current status of the field and some upcoming targets of interest that may lead to a safer next generation of analgesics. LINKED ARTICLES: This article is part of a themed issue on Advances in Opioid Pharmacology at the Time of the Opioid Epidemic. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v180.7/issuetoc.

The natural product argentatin C attenuates postoperative pain via inhibition of voltage-gated sodium and T-type voltage-gated calcium channels.

BACKGROUND AND PURPOSE: Postoperative pain occurs in as many as 70% of surgeries performed worldwide. Postoperative pain management still relies on opioids despite their negative consequences, resulting in a public health crisis. Therefore, it is important to develop alternative therapies to treat chronic pain. Natural products derived from medicinal plants are potential sources of novel biologically active compounds for development of safe analgesics. In this study, we screened a library of natural products to identify small molecules that target the activity of voltage-gated sodium and calcium channels that have important roles in nociceptive sensory processing. EXPERIMENTAL APPROACH: Fractions derived from the Native American medicinal plant, Parthenium incanum, were assessed using depolarization-evoked calcium influx in rat dorsal root ganglion (DRG) neurons. Further separation of these fractions yielded a cycloartane-type triterpene identified as argentatin C, which was additionally evaluated using whole-cell voltage and current-clamp electrophysiology, and behavioural analysis in a mouse model of postsurgical pain. KEY RESULTS: Argentatin C blocked the activity of both voltage-gated sodium and low-voltage-activated (LVA) calcium channels in calcium imaging assays. Docking analysis predicted that argentatin C may bind to NaV 1.7-1.9 and CaV 3.1-3.3 channels. Furthermore, argentatin C decreased Na+ and T-type Ca2+ currents as well as excitability in rat and macaque DRG neurons, and reversed mechanical allodynia in a mouse model of postsurgical pain. CONCLUSION AND IMPLICATIONS: These results suggest that the dual effect of argentatin C on voltage-gated sodium and calcium channels supports its potential as a novel treatment for painful conditions.

Discovery of κ Opioid Receptor (KOR)-Selective d-Tetrapeptides with Improved In Vivo Antinociceptive Effect after Peripheral Administration.

Peripherally active tetrapeptides as selective κ opioid receptor (KOR) agonists have been prepared in good overall yields and high purity following solid-phase peptide synthesis via Fmoc protection strategy. Structural modifications at the first and second position of the lead compound FF(d-Nle)R-NH2 (FE200041) were contemplated with aromatic side chains containing d-amino acids, such as (d)-pF-Phe, (d)-mF-Phe, (d)-oF-Phe, which led to highly selective and efficacious KOR agonists endowed with strong antinociceptive activity in vivo following intravenous (i.v.) and subcutaneous (s.c.) administration in the tail flick and formalin tests. These results suggest potential clinical applications in the treatment of neuropathic and inflammatory pain.

Inhibition of HSP90 Preserves Blood-Brain Barrier Integrity after Cortical Spreading Depression.

Cortical spreading depression (CSD) is a pathophysiological mechanism underlying headache disorders, including migraine. Blood-brain barrier (BBB) permeability is increased during CSD. Recent papers have suggested that heat shock proteins (HSP) contribute to the integrity of the blood-brain barrier. In this study, the possible role of HSP90 in CSD-associated blood-brain barrier leak at the endothelial cell was investigated using an in vitro model, for the blood-endothelial barrier (BEB), and an in vivo model with an intact BBB. We measured barrier integrity using trans endothelial electric resistance (TEER) across a monolayer of rodent brain endothelial cells (bEnd.3), a sucrose uptake assay, and in situ brain perfusion using female Sprague Dawley rats. CSD was induced by application of 60 mM KCl for 5 min in in vitro experiments or cortical injection of KCl (1 M, 0.5 µL) through a dural cannula in vivo. HSP90 was selectively blocked by 17-AAG. Our data showed that preincubation with 17-AAG (1 µM) prevented the reduction of TEER values caused by the KCl pulse on the monolayer of bEnd.3 cells. The elevated uptake of 14C-sucrose across the same endothelial monolayer induced by the KCl pulse was significantly reduced after preincubation with HSP90 inhibitor. Pre-exposure to 17-AAG significantly mitigated the transient BBB leak after CSD induced by cortical KCl injection as determined by in situ brain perfusion in female rats. Our results demonstrated that inhibition of HSP90 with the selective agent 17-AAG reduced CSD-associated BEB/BBB paracellular leak. Overall, this novel observation supports HSP90 inhibition mitigates KCl-induced BBB permeability and suggests the development of new therapeutic approaches targeting HSP90 in headache disorders.

Extracellular Alterations in pH and K+ Modify the Murine Brain Endothelial Cell Total and Phospho-Proteome.

Pathologies of the blood-brain barrier (BBB) have been linked to a multitude of central nervous system (CNS) disorders whose pathology is poorly understood. Cortical spreading depression (CSD) has long been postulated to be involved in the underlying mechanisms of these disease states, yet a complete understanding remains elusive. This study seeks to utilize an in vitro model of the blood-brain barrier (BBB) with brain endothelial cell (b.End3) murine endothelioma cells to investigate the role of CSD in BBB pathology by characterizing effects of the release of major pronociceptive substances into the extracellular space of the CNS. The application of trans-endothelial electrical resistance (TEER) screening, transcellular uptake, and immunoreactive methods were used in concert with global proteome and phospho-proteomic approaches to assess the effect of modeled CSD events on the modeled BBB in vitro. The findings demonstrate relocalization and functional alteration to proteins associated with the actin cytoskeleton and endothelial tight junctions. Additionally, unique pathologic mechanisms induced by individual substances released during CSD were found to have unique phosphorylation signatures in phospho-proteome analysis, identifying Zona Occludins 1 (ZO-1) as a possible pathologic "checkpoint" of the BBB. By utilizing these phosphorylation signatures, possible novel diagnostic methods may be developed for CSD and warrants further investigation.

Age-Induced Changes in µ-Opioid Receptor Signaling in the Midbrain Periaqueductal Gray of Male and Female Rats.

Opioids have decreased analgesic potency (but not efficacy) in aged rodents compared with adults; however, the neural mechanisms underlying this attenuated response are not yet known. The present study investigated the impact of advanced age and biological sex on opioid signaling in the ventrolateral periaqueductal gray (vlPAG) in the presence of chronic inflammatory pain. Assays measuring µ-opioid receptor (MOR) radioligand binding, GTPγS binding, receptor phosphorylation, cAMP inhibition, and regulator of G-protein signaling (RGS) protein expression were performed on vlPAG tissue from adult (2-3 months) and aged (16-18 months) male and female rats. Persistent inflammatory pain was induced by intraplantar injection of complete Freund's adjuvant (CFA). Adult males exhibited the highest MOR binding potential (BP) and highest G-protein activation (activation efficiency ratio) in comparison to aged males and females (adult and aged). No impact of advanced age or sex on MOR phosphorylation state was observed. DAMGO-induced cAMP inhibition was highest in the vlPAG of adult males compared with aged males and females (adult and aged). vlPAG levels of RGS4 and RGS9-2, critical for terminating G-protein signaling, were assessed using RNAscope. Adult rats (both males and females) exhibited lower levels of vlPAG RGS4 and RGS9-2 mRNA expression compared with aged males and females. The observed age-related reductions in vlPAG MOR BP, G-protein activation efficiency, and cAMP inhibition, along with the observed age-related increases in RGS4 and RGS9-2 vlPAG expression, provide potential mechanisms whereby the potency of opioids is decreased in the aged population.SIGNIFICANCE STATEMENTOpioids have decreased analgesic potency (but not efficacy) in aged rodents compared with adults; however, the neural mechanisms underlying this attenuated response are not yet known. In the present study, we observed age-related reductions in ventrolateral periaqueductal gray (vlPAG) µ-opioid receptor (MOR) binding potential (BP), G-protein activation efficiency, and cAMP inhibition, along with the observed age-related increases in regulator of G-protein signaling (RGS)4 and RGS9-2 vlPAG expression, providing potential mechanisms whereby the potency of opioids is decreased in the aged population. These coordinated decreases in opioid receptor signaling may explain the previously reported reduced potency of opioids to produce pain relief in females and aged rats.

Design and Synthesis of Brain Penetrant Glycopeptide Analogues of PACAP With Neuroprotective Potential for Traumatic Brain Injury and Parkinsonism.

There is an unmet clinical need for curative therapies to treat neurodegenerative disorders. Most mainstay treatments currently on the market only alleviate specific symptoms and do not reverse disease progression. The Pituitary adenylate cyclase-activating polypeptide (PACAP), an endogenous neuropeptide hormone, has been extensively studied as a potential regenerative therapeutic. PACAP is widely distributed in the central nervous system (CNS) and exerts its neuroprotective and neurotrophic effects via the related Class B GPCRs PAC1, VPAC1, and VPAC2, at which the hormone shows roughly equal activity. Vasoactive intestinal peptide (VIP) also activates these receptors, and this close analogue of PACAP has also shown to promote neuronal survival in various animal models of acute and progressive neurodegenerative diseases. However, PACAP's poor pharmacokinetic profile (non-linear PK/PD), and more importantly its limited blood-brain barrier (BBB) permeability has hampered development of this peptide as a therapeutic. We have demonstrated that glycosylation of PACAP and related peptides promotes penetration of the BBB and improves PK properties while retaining efficacy and potency in the low nanomolar range at its target receptors. Furthermore, judicious structure-activity relationship (SAR) studies revealed key motifs that can be modulated to afford compounds with diverse selectivity profiles. Most importantly, we have demonstrated that select PACAP glycopeptide analogues (2LS80Mel and 2LS98Lac) exert potent neuroprotective effects and anti-inflammatory activity in animal models of traumatic brain injury and in a mild-toxin lesion model of Parkinson's disease, highlighting glycosylation as a viable strategy for converting endogenous peptides into robust and efficacious drug candidates.

Correction: Tanguturi et al. Discovery of Novel Delta Opioid Receptor (DOR) Inverse Agonist and Irreversible (Non-Competitive) Antagonists. Molecules 2021, 26, 6693.

There was an error in the original publication [...].

Antagonism of the mu-delta opioid receptor heterodimer enhances opioid antinociception by activating Src and calcium/calmodulin-dependent protein kinase II signaling.

ABSTRACT: The opioid receptors are important regulators of pain, reward, and addiction. Limited evidence suggests the mu and delta opioid receptors form a heterodimer (MDOR), which may act as a negative feedback brake on opioid-induced analgesia. However, evidence for the MDOR in vivo is indirect and limited, and there are few selective tools available. We recently published the first MDOR-selective antagonist, D24M, allowing us to test the role of the MDOR in mice. We thus cotreated CD-1 mice with D24M and opioids in tail flick, paw incision, and chemotherapy-induced peripheral neuropathy pain models. D24M treatment enhanced oxymorphone antinociception in all models by 54.7% to 628%. This enhancement could not be replicated with the mu and delta selective antagonists CTAP, naltrindole, and naloxonazine, and D24M had a mild transient effect in the rotarod test, suggesting this increase is selective to the MDOR. However, D24M had no effect on morphine or buprenorphine, suggesting that only specific opioids interact with the MDOR. To find a mechanism, we performed phosphoproteomic analysis on brainstems of mice. We found that the kinases Src and CaMKII were repressed by oxymorphone, which was restored by D24M. We were able to confirm the role of Src and CaMKII in D24M-enhanced antinociception using small molecule inhibitors (KN93 and Src-I1). Together, these results provide direct in vivo evidence that the MDOR acts as an opioid negative feedback brake, which occurs through the repression of Src and CaMKII signal transduction. These results further suggest that MDOR antagonism could be a means to improve clinical opioid therapy.