Repeated administration of opioids such as morphine and fentanyl activates glial cells, leading to the release of pro-inflammatory molecules through secretory pathways, including theNLRP3 inflammasome. NLRP3 activation is thought to contribute to neuroinflammation, opioid tolerance, and opioid-induced hyperalgesia (OIH), serving as a potential mechanism behind the reduced effectiveness and adverse consequences of long-term opioid analgesic therapy. The present study examined the influence of NLRP3 on neurochemical markers of inflammation and synaptic plasticity in the brain, as well as behavioral and physiological responses to morphine and fentanyl in C57BL/6J mice. Treatment with morphine or fentanyl, alone or combined, produced dose-dependent antinociception, respiratory depression, and psychostimulatory behavior while significantly increasing inflammatory cytokines IL-1β, IL-6, IL-18, and TNF-α, along with chemokines MCP-1 and RANTES. This response was linked to higher expressed levels of NLRP3, MAPKs, JNK and p38, and the transcription factor NF-kB, while reducing synaptic plasticity markers NMDAR, AMPA, and PSD-95. In animals treated with the selective NLRP3 inhibitor MCC950 alongside opioid exposure, levels of IL-6, IL-18, TNF-α, and NLRP3 in the brain decreased, and PSD-95 levels were restored. Behaviorally, MCC950 potentiated opioid-induced antinociception, respiratory depression, hyperlocomotion, and the rewarding effects of morphine in the conditioned place preference test, although it mitigated morphine acute antinociceptive tolerance and OIH. In summary, these initial results indicate that the NLRP3 pathway influences the release of opioid-related inflammatory molecules, affecting behaviors that increase the risk of opioid overdose.

HIV-Tat and morphine synergistically exacerbate neuroinflammation and DNA damage, yet the mechanisms linking the DNA damage response (DDR), chromatin remodeling, and innate immune activation remain poorly understood. Here, we investigated how HIV-Tat and morphine alter DNA repair dynamics and cGAS–STING–AIM2 inflammasome signaling in microglia and cortical tissue and evaluated the efficacy of dimethyl fumarate (DMF) in mitigating these effects. HMC3 microglial cells were exposed to HIV-Tat (50 ng/mL) and morphine (10 µM) for 24 h or 5 days, with H2A.X knockdown performed prior to treatment. In vivo, doxycycline (DOX)-induced Tat transgenic mice received escalating morphine doses for 4 days, followed by DMF (30 mg/kg, i.p.) for 7 days. Postmortem frontal cortical tissues from HIV-positive and matched HIV-negative donors were analyzed for validation. Behavioral testing, immunostaining, qPCR, and Western blotting assessed DDR markers, KAT5 acetylation, histone modifications, and innate immune mediators. HIV-Tat and morphine significantly enhanced DDR activation in vitro and in vivo, as indicated by increased ATM and γH2AX and elevated KAT5 acetylation. Large Organized Chromatin Lysine (K) domains (LOCK) displayed reduced H3K9me3 and increased H3K9ac, reflecting chromatin relaxation and disrupted epigenetic control over DDR and inflammation. DOX+Morphine treatment upregulated ERCC1 and ERCC8 but downregulated ERCC2, ERCC3, and ERCC6, demonstrating impaired nucleotide excision repair, and these changes were closely associated with cGAS–STING activation and AIM2/NLRP3 inflammasome induction. H2A.X knockdown or DMF treatment suppressed ATM phosphorylation, restored H3K9me3, normalized ERCC expression, and attenuated inflammasome activation. DMF also rescued HIV-Tat ± morphine-induced deficits in novel object recognition. Postmortem cortical tissue confirmed γH2AX accumulation, reduced ERCC2/ERCC3, and altered histone signatures consistent with experimental findings. Together, these results demonstrate that HIV-Tat and morphine cooperatively disrupt LOCK chromatin and DDR through the ATM/γH2AX axis, and that DMF reverses these molecular and functional abnormalities, highlighting its therapeutic potential in mitigating HIV- and opioid-induced neuroinflammation and cognitive decline.

ABSTRACT:Noxious cold sensation is commonly associated with peripheral neuropathies; however, there has been limited progress in understanding the mechanism. Here, we identify a novel role for peripherally expressed kappa opioid receptors [KORs] in noxious cold hypersensitivity in a mouse model of chemotherapy. In this model, we show that oxaliplatin-induced cold hypersensitivity is attenuated using norbinaltorphimine (NorBNI, KOR antagonist (10 mg/kg, i.p.) in both male and female mice. To further examine the role of KORs in cold hypersensitivity, we show that activation of KOR with U50,488 (KOR agonist, 5 mg/kg i.p.) significantly increases the number of jumps on a cold plate at 3°C as compared to controls, which is attenuated by KOR antagonist NorBNI. We have determined that this effect is mediated through peripheral KORs using both pharmacology and a peripherally restricted KOR agonist, ff(nle)r-NH2. Using a conditional knockout mouse model of KOR in the dorsal root ganglia (using PirtCre;Oprk fl/fl ), we see reduced U50488-induced noxious cold hypersensitivity as compared to controls in male and female mice, confirming the role of KORs in the dorsal root ganglia in mediating cold hypersensitivity. To confirm the role of peripheral KORs in oxaliplatin-induced cold allodynia, we use the PirtCre;Oprk fl/fl in our oxaliplatin-induced cold allodynia model. Here, we show that in the absence of KORs in dorsal root ganglia, oxaliplatin-induced cold hypersensitivity is attenuated in male and female mice. Overall, our data suggest that peripheral KORs modulate cold hypersensitivity in a mouse model of chemotherapy.

Background/Objectives: Neuropathic pain remains a significant clinical challenge, with current treatments often providing inadequate relief and adverse effects. Sigma receptors (SRs) modulate nociception and have emerged as potential therapeutic targets for neuropathic pain. Although putative sigma-1 receptor (S1R) ligands have demonstrated analgesic efficacy in preclinical models, their in vivo efficacy and safety profiles require further clarification. Methods: Analogs of well-known selective S1R ligand UVM147 were synthesized using 3-component Ugi reactions and examined in vitro for receptor affinity in radioligand competition binding assays and in vivo with mouse models of neuropathic and inflammatory pain and adverse effects. Results: Three novel heterocyclic compounds (RO-4-3, RO-5-3, and RO-7-3) displayed in vitro nanomolar affinity with varying selectivity for both SR subtypes (S1R and S2R). When screened in vivo at a dose of 30 mg/kg s.c. in mice first subjected to chronic constriction injury (CCI), RO-5-3 and RO-7-3 possessed anti-allodynic potential, while UVM147 was inactive. Upon full characterization, RO-5-3 significantly attenuated mechanical allodynia in a dose-dependent manner, while RO-7-3 was ineffective at higher doses. Both compounds dose-dependently attenuated nociceptive behaviors in the mouse formalin assay. RO-5-3 induced mild respiratory depression without impairing locomotor activity, whereas RO-7-3 caused transient respiratory depression and locomotor impairment. Additionally, RO-5-3, but not RO-7-3, induced conditioned place aversion consistent with potential S2R involvement. Conclusions: RO-5-3 exerts antinociceptive and anti-allodynic effects with minimal adverse behavioral effects, supporting the role of SRs in pain modulation. These results add to growing evidence supporting the development of SR ligands as efficacious therapeutics for neuropathic pain with fewer clinical liabilities.

Chronic pain affects hundreds of millions of people and remains poorly managed, as the most effective drugs, including opioids, carry side effects that limit long-term use. Because inflammation drives both the initiation and maintenance of chronic pain, anti-inflammatory mechanisms are an attractive analgesic target. We investigated human alpha-1 antitrypsin (hAAT), a serine proteinase inhibitor with potent anti-inflammatory activity and established protection across disease models, as a candidate analgesic, using Aralast NP®, a clinical-grade, already-approved formulation that makes findings directly translatable. Using calcium imaging and patch-clamp electrophysiology in mouse dorsal root ganglion (DRG) neurons, we found that hAAT reduced activation of low-voltage-activated Ca2+ channels and dampened intrinsic excitability. Veratridine-evoked Ca2+ responses, a sodium-channel-dependent readout of nociceptor activity, were suppressed by hAAT to a degree comparable to the selective sodium channel inhibitors ProTx-II (NaV1.7) and VX-548 (NaV1.8), driven by loss of the nociceptor-associated response profiles. In vivo, hAAT decreased pain sensitivity and pain-associated behaviors in both inflammatory and neuropathic models. Together, these findings reveal a mechanism by which hAAT suppresses nociceptor activity and position Aralast NP® as a safe, effective candidate for treating chronic pain.

Mitochondrial energy deficits play a central role in HIV-associated neurocognitive disorder (HAND). HIV disrupts cellular functions, including epigenetic modifications such as class III histone deacetylation mediated by sirtuins (SIRTs). However, the role of SIRTs in HAND pathogenesis remains unclear. We hypothesize that HIV alters mitochondrial biogenesis and energy homeostasis by modifying SIRT family members 1–7, contributing to HAND progression. To test this hypothesis, we examined postmortem frontal lobe brain tissue from people with HIV (PWH) and HIV-negative controls, focusing on epigenetic alterations in SIRTs 1–7, the energy sensor adenosine monophosphate-activated protein kinase (AMPK), the mitochondrial master regulator peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), and transcription factors such as mitochondrial transcription factor A (TFAM), nuclear respiratory factors 1 and 2 (NRF-1/2), and factors associated with oxidative phosphorylation (OXPHOS). Our analysis revealed a significant increase in AMPK, OXPHOS, and PGC-1α levels, alongside a decrease in TFAM levels in PWH brains compared to uninfected controls. NRF-1 was upregulated in mitochondria but downregulated in the cytoplasm, while NRF-2 exhibited the opposite trend in PWH compared to HIV-negative controls. The epigenetic signatures of SIRTs 1, 2, 3, 4, 6, and 7 were upregulated in PWH, while SIRT5 was downregulated compared to uninfected brain tissues. We exposed primary human astrocyte and microglial cultures to the HIV-1 transactivator of transcription (Tat) protein to identify the cell types involved. These studies confirmed that HIV-induced epigenetic modifications of SIRTs and mitochondrial impairments occurred in both astrocytes and microglia, highlighting the crucial role of SIRTs in HAND pathogenesis.

Morphine is a µ-opioid receptor (MOR) agonist and potent analgesic. However, it displays several side effects including respiratory depression and addiction. Here, we show that a single heavy atom replacement in the morphine core structure (O to CH 2 exchange in the E-ring) prepared through a 15-step total synthesis displays a different pharmacological profile. The total synthesis features an intramolecular inverse electron-demand Diels−Alder cycloaddition and a stereoselective Giese radical addition to construct a quaternary carbon center. Unlike morphine, where the (–)-morphine enantiomer binds the MOR, both enantiomers of this “carba” variant, which we have named carbamorphine, possess activity as agonists of the MOR. Cell-based functional assays show that (+)-carbamorphine shows reduced G-protein as well as β-arrestin efficacy at the MOR. In mouse behavioral assays, (+)-carbamorphine exhibits MOR-selective antinociception while showing reduced respiratory depression and a lack of conditioned place preference at supratherapeutic doses. Overall, through a net “single-atom” change (i.e., O to CH 2 ) in the morphine framework, different pharmacological profiles have been realized. This work provides a basis for additional syntheses and the study of morphine analogs that incorporate atom changes in the core framework.

Heterocyclic peptidomimetics are constrained compounds that mimic the biological efficacy of peptides while offering increased stability. We have previously generated a diazaheterocyclic peripherally selective, mixed-opioid agonist peptidomimetic that produced synergistic antinociception with decreased side effects. Working from two earlier templates, we report here the synthesis of 15 new diazaheterocyclic analogues. In vitro screening with radioligand competition binding assays and [35S]GTPγS assays demonstrated variable affinity for and activity at μ (MOR), δ (DOR), and κ (KOR) opioid receptors across the series, with three (2663-48, 2638-28 and 2638-33) displaying good affinity for DOR and/or KOR. All three compounds produced dose-dependent, opioid-receptor mediated antinociception in the mouse 55 °C warm-water tail-withdrawal and acetic-acid writhing assay, although a ratio of ED50 values in these assays suggested poor BBB penetration by 2638-33; results confirmed by testing with naloxone-methiodide. The data suggest these diazaheterocyclic mixed-activity, peripherally restricted opioid receptor agonists may hold potential as new, safer analgesics.

Chronic pain and opioid overdose deaths highlight the need for non-addictive analgesics with novel mechanisms. The δ opioid receptor (δOR) is a promising target, as it lacks the respiratory depression associated with µ opioid receptor (µOR) agonists. However, early δOR full agonists caused seizures, limiting their clinical use. Partial δOR agonists may offer more controlled receptor activation than full agonists, but their development has been hindered by uncertainty regarding the molecular mechanism of partial agonism. Here we show that C6-Quino, a bitopic ligand developed through structure-based design, acts as a selective δOR partial agonist. Functional studies reveal that C6-Quino shows differential activity at G-protein and arrestin pathways and interacts with the sodium binding pocket, confirmed through cryo-EM analysis. C6-Quino demonstrates oral activity, analgesic activity in chronic pain models without causing δOR-related seizures and µOR-related adverse effects which have limited opioid usage in recent times. This discovery outlines a new strategy for developing δOR-targeted analgesics and provides a framework for optimizing signaling profiles of other Class A GPCRs. δ-Opioid receptors (δOR) are promising targets for pain management with reduced side effects. Here, the authors use a structure-based approach to design and characterize C6-Quino, a selective δOR partial agonist, highlighting its potential therapeutic relevance.

Kappa opioid receptor (KOR) antagonists may have therapeutic potential to prevent stress-induced relapse in abstinent individuals with cocaine use disorder (CUD). The macrocyclic peptide [D-Trp]CJ-15,208 (cyclo[Phe-D-Pro-Phe-D-Trp]) is an orally bioavailable, brain–penetrant selective KOR antagonist that prevents stress-induced reinstatement of cocaine-seeking behavior in a mouse model of CUD. We synthesized and evaluated analogs of this lead compound with substitutions for the D-Trp residue to identify analogs that exhibit more potent central KOR antagonism following oral administration. The peptides were synthesized by a combination of solid phase and solution peptide synthetic methodologies, and their pharmacological activity was evaluated both in vitro (for KOR affinity, selectivity and antagonism) and in vivo (for antinociception and KOR antagonism), with promising analogs evaluated for their ability to prevent stress-induced reinstatement of cocaine-seeking behavior in the mouse conditioned place preference (CPP) assay. A variety of substituted D-Phe or modified D-Trp derivatives were tolerated by KOR with retention of significant KOR antagonism in vivo after oral administration. Macrocyclic peptide pretreatment, per os, significantly prevented stress-induced reinstatement of cocaine CPP at doses of 10 and 30 mg/kg of [D-Phe4]CJ-15,208, 4, and 30 mg/kg of [D-Trp(formamide)]CJ-15,208, 3, which are 6-fold and 2-fold lower, respectively, than that needed for {D-Trp]CJ-15,208.

Neuropeptide FF (NPFF) receptor antagonists prevent morphine-mediated antinociceptive tolerance, and compounds with dual mu opioid receptor (MOR) agonist and NPFF antagonist activity produce antinociception without tolerance. Compounds synthesized showed affinities in radioligand competition binding assays in the nM and µM range at the opioid and NPFF receptors, respectively, and displayed substitution-dependent functional profiles in the [35S]GTPγS functional assay. From six compounds screened in vivo for antinociception and ability to prevent NPFF-induced hyperalgesia in mouse warm water tail withdrawal tests, compound 22b produced dose-dependent MOR-mediated antinociception with an ED50 value (and 95% confidence interval) of 6.88 (4.71–9.47) nmol, i.c.v., and also prevented NPFF-induced hyperalgesia. Meanwhile, 22b did not demonstrate the respiratory depression, hyperlocomotion, or impaired intestinal transit of morphine. Moreover, repeated treatment with 22b produced a 1.6-fold rightward shift in antinociceptive dose response, significantly less acute antinociceptive tolerance than morphine. Evaluated for microsomal stability in vitro and in vivo pharmacokinetic profile, 22b showed suitable microsomal stability paired in vivo with a large apparent volume of distribution and a clearance smaller than the hepatic flow in rats, suggesting no extra-hepatic metabolism. In conclusion, the present study confirms that dual-action opioid–NPFF ligands may offer therapeutic promise as analgesics with fewer liabilities of use.

We present the synthesis and pharmacological characterization of 3'-iodobenzoylamido epoxymorphinans, with a saturated ring C and devoid of 14-OH, based on a dihydronormorphine backbone with various N-17 alkyl substitutions. All synthesized compounds were characterized pharmacologically in radioligand binding assays, [35S]GTPγS functional assays, and for antinociception in mice. Among these analogues, MP1202, a pan-opioid receptor analogue, was characterized further in β-arrestin1/2 studies, G-protein recruitment and Gα-subtype profiling, as well as by evaluation of opioid-mediated effects in mice. MP1202 exhibits potent antinociceptive effects in mice without causing typical opioid side effects, such as respiratory depression and physical dependence. However, it displays conditioned place preference and aversion behaviors similar to those of classical mu and kappa agonists.

Abstract ID 100583Poster Board 286The rising prevalence of chronic pain and overdose deaths from classical opioids targeting μ opioid receptor (μOR) have fueled a need for reliable long-term analgesics, which use different targets and mechanisms. The δ opioid receptor (δOR) has been considered an attractive alternative target for non-addictive analgesics, especially for its lack of constipation and respiratory depression and effectiveness in alleviating chronic pain. However, δOR activation was found to induce seizures, precluding clinical use of full δOR agonists. Partial agonists may offer a more controlled and gradual activation of a receptor compared to full agonists, however molecular mechanism of partial agonism has remained unclear, hindering the development of such ligands. Using a structure-based approach, we explored the engagement of the sodium binding pocket in δ opioid receptor (δOR) and identified the first selective δOR partial agonist, C6-Quino. Functional studies of C6 revealed that it displayed partial agonist activity at both G protein and arrestin pathways. Its interactions with the Na+ pocket were confirmed in the single particle cryo-EM structure coupled with molecular dynamics simulations. Finally, C6-Quino demonstrated favorable physiological properties conferring sufficient brain permeability, oral activity, and analgesic activity in multiple chronic pain models. Notably, neither μOR related locomotion interruption and respiratory depression nor δOR related convulsions were observed at analgesic doses of C6-Quino. As a fundamentally new approach on δOR, this methodology provides a blueprint toward the development of partial agonists as safe analgesics and acts as a generic method to optimize signaling profiles of other Class A GPCR’s as well.

Abstract ID 95191Poster Board 084Chronic pain is highly prevalent and is one of the most common reasons adults seek medical attention. Unfortunately, current therapeutics to treat this type of pain are limited due to low efficacy and/or high side effect profile. Delta opioid receptor (DOR) agonists have been found to be particularly effective at treating chronic pain and have anxiolytic and anti-depressant effects which are commonly comorbid with chronic pain. However, DOR agonists exhibit negative side effects like seizures and tolerance. To develop safe and effective DOR agonists, we need to understand how they bind and signal. We have used cryo-electron microscopy (cryoEM) to solve DOR-Gi1 structures of pharmacologically distinct agonists, including an endogenous peptide, a seizure-causing ligand, a seizure-free ligand which entered Phase 2 clinical trials, an antagonist, and a structure with a positive allosteric modulator. We have also solved the DOR-Gi1 structure of a newly developed DOR partial agonist which is effective at treating different types of pain in mice, including chronic pain, and has a limited side effect profile. Comparing cryoEM structures of DOR agonists with different signaling and behavioral profiles can guide the development of next-generation DOR agonists to treat chronic pain.This research was supported by the PhRMA foundation and NIH grant R01DA057790.

The mu opioid receptor (mu OR) is a target for clinically used analgesics. However, adverse effects, such as respiratory depression and physical dependence, necessitate the development of alternative treatments. Recently we reported a novel strategy to design functionally selective opioids by targeting the sodium binding allosteric site in mu OR with a supraspinally active analgesic named C6guano. Presently, to improve systemic activity of this ligand, we used structure-based design, identifying a new ligand named RO76 where the flexible alkyl linker and polar guanidine guano group is swapped with a benzyl alcohol, and the sodium site is targeted indirectly through waters. A cryoEM structure of RO76 bound to the mu OR-G(i) complex confirmed that RO76 interacts with the sodium site residues through a water molecule, unlike C6guano which engages the sodium site directly. Signaling assays coupled with APEX based proximity labeling show binding in the sodium pocket modulates receptor efficacy and trafficking. In mice, RO76 was systemically active in tail withdrawal assays and showed reduced liabilities compared to those of morphine. In summary, we show that targeting water molecules in the sodium binding pocket may be an avenue to modulate signaling properties of opioids, and which may potentially be extended to other G-protein coupled receptors where this site is conserved.

PRX-3140 is a partial agonist to the 5-hydroxytryptamine receptor 4 (5-HT4) and a ligand for the sigma-1 (S1R) and sigma-2 (S2R) receptors. Although few publications have inferred S1R agonists/antagonists modulate blood glucose, Di et.al (2017) reported S1R deficiency in knockout mice impacted regulation of the hypothalamic-pituitary-adrenocortical (HPA) axis, with a dexamethasone-induced reduction in level of corticosterone markedly attenuated in S1R −/− knockout mice, implicating S1R in feedback response to the HPA axis. The hypothesis that S1R deficiency causes down-regulation of the glucocorticoid receptor (GR) and attenuates GR-mediated feedback inhibition of HPA axis, as well as stress response of HPA axis, suggest that the inverse, the activation of S1R under normal conditions, may modulate glucocorticoid insulin suppression (as a direct S1R-GR effect) as well as cortisol levels (producing HPA axis feedback inhibition). In the present study, coadministration of 10 microM PRX-3140 with 100 nM cortisol significantly increased insulin release (to 74.8 ng/ml, P-value <0.0001). Similar effects were observed when cells were exposed to dexamethasone (Dex), with 10 microM PRX-3140 and 10 nM Dex producing 1.87-fold significantly more insulin than 10 nM Dex alone. Daily glucose concentrations in the 14-day clinical study ([NCT00384423][1]) of PRX-3140 demonstrate a reduction for 10 mg once-daily at days 1, 7, 10, and 15. Urine free cortisol levels at 10, 30, 100 and 200 mg dose levels of PRX-3140 demonstrated a larger reduction at 7 and 14 days compared to placebo. As an agonist of S1R that acts as a chaperone of GR, PRX-3140 has demonstrated GR modulating effects in INS-1 cells and in 14-day clinical studies in healthy adults with low incidence of side effects. The results of the present study suggest that S1R activation, with PRX-3140 and NP-18-2 S1R agonists, modulates glucocorticoid insulin suppression and cortisol levels. ### Competing Interest Statement JDT is an employee of Nanopharmaceutics, Inc., which holds rights in PRX-3140, NP-18-2 and NP-18-3. ### Clinical Trial NCT00384423 ### Funding Statement This study did not receive any funding ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Heartland Institutional Review Board & Privacy Board of Heartland IRB (Lenexa, KS) gave ethical approval for this work I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00384423&atom=%2Fmedrxiv%2Fearly%2F2024%2F12%2F12%2F2024.12.04.24318244.atom

CycloAnt is an opioid peptide that produces potent and efficacious antinociception with significantly reduced side effects upon systemic administration in mice. To verify its CNS-mediated antinociception, we determined its binding affinity at the opioid receptors, its proteolytic stability in mouse serum, metabolic stability in mouse liver microsomes, and pharmacokinetics in mice. CycloAnt exhibited stability toward proteolytic degradation in serum and resistance against metabolism mediated by cytochrome P450 enzymes (CYP450s) and UDP-glucuronosyl transferases (UGTs) in mouse liver microsomes. A pharmacokinetic study of CycloAnt in mice confirmed that CycloAnt crossed the blood-brain barrier (BBB) with a brain-to-plasma ratio of 11.5%, a high extent of BBB transport for a peptide. To elucidate the structural basis underlying its BBB penetration, we investigated its conformation in water and DMSO using 1H NMR spectroscopy. The results show that CycloAnt displays an extended conformation in water with most amide NHs being exposed, while in less polar DMSO, it adopts a compact conformation with all amide NHs locked in intramolecular hydrogen bonds. The chameleonic property helps CycloAnt permeate the BBB.

Perturbation of dopamine transmission has been implicated as a contributing factor in HIV-1 associated neurocognitive disorders with concurrent methamphetamine (METH) abuse. We have demonstrated that the HIV-1 protein, transactivator of transcription (Tat), decreases dopamine transport through inhibition of vesicular monoamine transporter2 (VMAT2). This study determined the effects of Tat protein on METH-inhibited VMAT2 function and METH-conditioned place preference (CPP). In vitro exposure of isolated mouse whole brain vesicles to recombinant Tat1-86 or METH displayed a concentration-dependent inhibition of the vesicular [3H]Dopamine uptake, in which a combination of Tat and METH induced a greater reduction of dopamine uptake compared to Tat or METH alone. In vivo, the maximal velocity (Vmax) of vesicular [3H]Dopamine uptake was decreased in inducible Tat transgenic (iTat-tg) mice harvested after treatment with either 21-day doxycycline (Dox) or 14-day METH (3 mg/kg, i.p., daily), whereas these mice treated with both Dox and METH displayed an additive reduction of the Vmax compared to either Tat or METH alone. Moreover, Dox-induced Tat expression increased METH-CPP in an exposure-dependent manner, with iTat-tg mice demonstrating a 2.3-fold potentiation of METH-CPP compared with Tat null control mice upon administration of Dox for 14 days. Furthermore, a 7-day administration of Dox reinstated extinguished METH-CPP. Collectively, these results suggest a synergistic effect of Tat protein and METH on inhibition of VMAT2-mediated DA transport, potentially contributing to potentiation of METH-CPP in iTat-tg mice.

The µ-opioid receptor (µOR) is a well-established target for analgesia1, yet conventional opioid receptor agonists cause serious adverse effects, notably addiction and respiratory depression. These factors have contributed to the current opioid overdose epidemic driven by fentanyl2, a highly potent synthetic opioid. µOR negative allosteric modulators (NAMs) may serve as useful tools in preventing opioid overdose deaths, but promising chemical scaffolds remain elusive. Here we screened a large DNA-encoded chemical library against inactive µOR, counter-screening with active, G-protein and agonist-bound receptor to 'steer' hits towards conformationally selective modulators. We discovered a NAM compound with high and selective enrichment to inactive µOR that enhances the affinity of the key opioid overdose reversal molecule, naloxone. The NAM works cooperatively with naloxone to potently block opioid agonist signalling. Using cryogenic electron microscopy, we demonstrate that the NAM accomplishes this effect by binding a site on the extracellular vestibule in direct contact with naloxone while stabilizing a distinct inactive conformation of the extracellular portions of the second and seventh transmembrane helices. The NAM alters orthosteric ligand kinetics in therapeutically desirable ways and works cooperatively with low doses of naloxone to effectively inhibit various morphine-induced and fentanyl-induced behavioural effects in vivo while minimizing withdrawal behaviours. Our results provide detailed structural insights into the mechanism of negative allosteric modulation of the µOR and demonstrate how this can be exploited in vivo.