Positive allosteric mechanisms of adenosine A1 receptor-mediated analgesia.

The adenosine A1 receptor (A1R) is a promising therapeutic target for non-opioid analgesic agents to treat neuropathic pain1,2. However, development of analgesic orthosteric A1R agonists has failed because of a lack of sufficient on-target selectivity as well as off-tissue adverse effects3. Here we show that [2-amino-4-(3,5-bis(trifluoromethyl)phenyl)thiophen-3-yl)(4-chlorophenyl)methanone] (MIPS521), a positive allosteric modulator of the A1R, exhibits analgesic efficacy in rats in vivo through modulation of the increased levels of endogenous adenosine that occur in the spinal cord of rats with neuropathic pain. We also report the structure of the A1R co-bound to adenosine, MIPS521 and a Gi2 heterotrimer, revealing an extrahelical lipid-detergent-facing allosteric binding pocket that involves transmembrane helixes 1, 6 and 7. Molecular dynamics simulations and ligand kinetic binding experiments support a mechanism whereby MIPS521 stabilizes the adenosine-receptor-G protein complex. This study provides proof of concept for structure-based allosteric drug design of non-opioid analgesic agents that are specific to disease contexts.

Dendritic function of tau mediates amyloid-beta toxicity in Alzheimer's disease mouse models.

Alzheimer's disease (AD) is characterized by amyloid-beta (Abeta) and tau deposition in brain. It has emerged that Abeta toxicity is tau dependent, although mechanistically this link remains unclear. Here, we show that tau, known as axonal protein, has a dendritic function in postsynaptic targeting of the Src kinase Fyn, a substrate of which is the NMDA receptor (NR). Missorting of tau in transgenic mice expressing truncated tau (Deltatau) and absence of tau in tau(-/-) mice both disrupt postsynaptic targeting of Fyn. This uncouples NR-mediated excitotoxicity and hence mitigates Abeta toxicity. Deltatau expression and tau deficiency prevent memory deficits and improve survival in Abeta-forming APP23 mice, a model of AD. These deficits are also fully rescued with a peptide that uncouples the Fyn-mediated interaction of NR and PSD-95 in vivo. Our findings suggest that this dendritic role of tau confers Abeta toxicity at the postsynapse with direct implications for pathogenesis and treatment of AD.

Spinal glycinergic currents are reduced in a rat model of neuropathic pain following partial nerve ligation but not chronic constriction injury.

Animal models have consistently indicated that central sensitization and the development of chronic neuropathic pain are linked to changes to inhibitory signaling in the dorsal horn of the spinal cord. However, replication of data investigating the cellular mechanisms that underlie these changes remains a challenge and there is still a lack of understanding about what aspects of spinal inhibitory transmission most strongly contribute to the disease. Here, we compared the effect of two different sciatic nerve injuries commonly used to generate rodent models of neuropathic pain on spinal glycinergic signaling. Using whole cell patch-clamp electrophysiology in spinal slices, we recorded from neurons in the lamina II of the dorsal horn and evoked inhibitory postsynaptic currents with a stimulator in lamina III, where glycinergic cell bodies are concentrated. We found that glycine inputs onto radial neurons were reduced following partial nerve ligation (PNL) of the sciatic nerve, consistent with a previous report. However, this finding was not replicated in animals that underwent chronic constriction injury (CCI) to the same nerve region. To limit the between-experiment variability, we kept the rat species, sex, and age consistent and had a single investigator carry out the surgeries. These data show that PNL and CCI cause divergent spinal signaling outcomes in the cord and add to the body of evidence suggesting that treatments for neuropathic pain should be triaged according to nerve injury or cellular dysfunction rather than the symptoms of the disease.NEW & NOTEWORTHY Neuropathic pain models are used in preclinical research to investigate the mechanisms underlying allodynia, a common symptom of neuropathic pain, and to test, develop, and validate therapies for persistent pain. We demonstrate that a glycinergic dysfunction is consistently associated with partial nerve ligation but not the chronic constriction injury model. This suggests that the cellular effects produced by each injury are distinct and that data from different neuropathic pain models should be considered separately.

Peripheral Administration of Selective Glycine Transporter-2 Inhibitor, Oleoyl-D-Lysine, Reverses Chronic Neuropathic Pain but Not Acute or Inflammatory Pain in Male Mice.

Aberrations in spinal glycinergic signaling are a feature of pain chronification. Normalizing these changes by inhibiting glycine transporter (GlyT)-2 is a promising treatment strategy. However, existing GlyT2 inhibitors (e.g., ORG25543) are limited by narrow therapeutic windows and severe dose-limiting side effects, such as convulsions, and are therefore poor candidates for clinical development. Here, intraperitoneally administered oleoyl-D-lysine, a lipid-based GlyT2 inhibitor, was characterized in mouse models of acute (hot plate), inflammatory (complete Freund's adjuvant), and chronic neuropathic (chronic constriction injury) pain. Side effects were also assessed on a numerical rating score, convulsions score, for motor incoordination (rotarod), and for respiratory depression (whole body plethysmography). Oleoyl-D-lysine produced near complete antiallodynia for chronic neuropathic pain, but no antiallodynia/analgesia in inflammatory or acute pain. No side effects were seen at the peak analgesic dose, 30 mg/kg. Mild side effects were observed at the highest dose, 100 mg/kg, on the numerical rating score, but no convulsions. These results contrasted markedly with ORG25543, which reached less than 50% reduction in allodynia score only at the lethal/near-lethal dose of 50 mg/kg. At this dose, ORG25543 caused maximal side effects on the numerical rating score and severe convulsions. Oleoyl-D-lysine (30 mg/kg) did not cause any respiratory depression, a problematic side effect of opiates. These results show the safe and effective reversal of neuropathic pain in mice by oleoyl-D-lysine and provide evidence for a distinct role of glycine in chronic pain over acute or short-term pain conditions. SIGNIFICANCE STATEMENT: Partially inhibiting glycine transporter (GlyT)-2 can alleviate chronic pain by restoring lost glycinergic function. Novel lipid-based GlyT2 inhibitor ol-D-lys is safe and effective in alleviating neuropathic pain, but not inflammatory or acute pain. Clinical application of GlyT2 inhibitors may be better suited to chronic neuropathic pain over other pain aetiologies.

A tetrapeptide class of biased analgesics from an Australian fungus targets the µ-opioid receptor.

An Australian estuarine isolate of Penicillium sp. MST-MF667 yielded 3 tetrapeptides named the bilaids with an unusual alternating LDLD chirality. Given their resemblance to known short peptide opioid agonists, we elucidated that they were weak (Ki low micromolar) µ-opioid agonists, which led to the design of bilorphin, a potent and selective µ-opioid receptor (MOPr) agonist (Ki 1.1 nM). In sharp contrast to all-natural product opioid peptides that efficaciously recruit ß-arrestin, bilorphin is G protein biased, weakly phosphorylating the MOPr and marginally recruiting ß-arrestin, with no receptor internalization. Importantly, bilorphin exhibits a similar G protein bias to oliceridine, a small nonpeptide with improved overdose safety. Molecular dynamics simulations of bilorphin and the strongly arrestin-biased endomorphin-2 with the MOPr indicate distinct receptor interactions and receptor conformations that could underlie their large differences in bias. Whereas bilorphin is systemically inactive, a glycosylated analog, bilactorphin, is orally active with similar in vivo potency to morphine. Bilorphin is both a unique molecular tool that enhances understanding of MOPr biased signaling and a promising lead in the development of next generation analgesics.

Intrinsic Efficacy of Opioid Ligands and Its Importance for Apparent Bias, Operational Analysis, and Therapeutic Window.

Evidence from several novel opioid agonists and knockout animals suggests that improved opioid therapeutic window, notably for analgesia versus respiratory depression, is a result of ligand bias downstream of activation of the µ-opioid receptor (MOR) toward G protein signaling and away from other pathways, such as arrestin recruitment. Here, we argue that published claims of opioid bias based on application of the operational model of agonism are frequently confounded by failure to consider the assumptions of the model. These include failure to account for intrinsic efficacy and ceiling effects in different pathways, distortions introduced by analysis of amplified (G protein) versus linear (arrestin) signaling mechanisms, and nonequilibrium effects in a dynamic signaling cascade. We show on both theoretical and experimental grounds that reduced intrinsic efficacy that is unbiased across different downstream pathways, when analyzed without due considerations, does produce apparent but erroneous MOR ligand bias toward G protein signaling, and the weaker the G protein partial agonism is the greater the apparent bias. Experimentally, such apparently G protein-biased opioids have been shown to exhibit low intrinsic efficacy for G protein signaling when ceiling effects are properly accounted for. Nevertheless, such agonists do display an improved therapeutic window for analgesia versus respiratory depression. Reduced intrinsic efficacy for G proteins rather than any supposed G protein bias provides a more plausible, sufficient explanation for the improved safety. Moreover, genetic models of G protein-biased opioid receptors and replication of previous knockout experiments suggest that reduced or abolished arrestin recruitment does not improve therapeutic window for MOR-induced analgesia versus respiratory depression. SIGNIFICANCE STATEMENT: Efforts to improve safety of µ-opioid analgesics have focused on agonists that show signaling bias for the G protein pathway versus other signaling pathways. This review provides theoretical and experimental evidence showing that failure to consider the assumptions of the operational model can lead to large distortions and overestimation of actual bias. We show that low intrinsic efficacy is a major determinant of these distortions, and pursuit of appropriately reduced intrinsic efficacy should guide development of safer opioids.

Endosomal signaling of the receptor for calcitonin gene-related peptide mediates pain transmission.

G protein-coupled receptors (GPCRs) are considered to function primarily at the plasma membrane, where they interact with extracellular ligands and couple to G proteins that transmit intracellular signals. Consequently, therapeutic drugs are designed to target GPCRs at the plasma membrane. Activated GPCRs undergo clathrin-dependent endocytosis. Whether GPCRs in endosomes control pathophysiological processes in vivo and are therapeutic targets remains uncertain. We investigated the contribution of endosomal signaling of the calcitonin receptor-like receptor (CLR) to pain transmission. Calcitonin gene-related peptide (CGRP) stimulated CLR endocytosis and activated protein kinase C (PKC) in the cytosol and extracellular signal regulated kinase (ERK) in the cytosol and nucleus. Inhibitors of clathrin and dynamin prevented CLR endocytosis and activation of cytosolic PKC and nuclear ERK, which derive from endosomal CLR. A cholestanol-conjugated antagonist, CGRP8-37, accumulated in CLR-containing endosomes and selectively inhibited CLR signaling in endosomes. CGRP caused sustained excitation of neurons in slices of rat spinal cord. Inhibitors of dynamin, ERK, and PKC suppressed persistent neuronal excitation. CGRP8-37-cholestanol, but not unconjugated CGRP8-37, prevented sustained neuronal excitation. When injected intrathecally to mice, CGRP8-37-cholestanol inhibited nociceptive responses to intraplantar injection of capsaicin, formalin, or complete Freund's adjuvant more effectively than unconjugated CGRP8-37 Our results show that CLR signals from endosomes to control pain transmission and identify CLR in endosomes as a therapeutic target for pain. Thus, GPCRs function not only at the plasma membrane but also in endosomes to control complex processes in vivo. Endosomal GPCRs are a drug target that deserve further attention.

Electrophysiological Actions of N/OFQ.

Whilst the nociceptin/orphanin FQ (N/OFQ) receptor (NOP) has similar intracellular coupling mechanisms to opioid receptors, it has distinct modulatory effects on physiological functions such as pain. These actions range from agonistic to antagonistic interactions with classical opioids within the spinal cord and brain, respectively. Understanding the electrophysiological actions of N/OFQ has been crucial in ascertaining the mechanisms by which these agonistic and antagonistic interactions occur. These similarities and differences between N/OFQ and opioids are due to the relative location of NOP versus opioid receptors on specific neuronal elements within these CNS regions. These mechanisms result in varied cellular actions including postsynaptic modulation of ion channels and presynaptic regulation of neurotransmitter release.

Correction to: Electrophysiological Actions of N/OFQ.

In the last paragraph of Sect. 2.1.2 on line 3 the word 'off-cells' is misspelt. It should be 'on-cells'.

Substance P and dopamine interact to modulate the distribution of delta-opioid receptors on cholinergic interneurons in the striatum.

It has been recently demonstrated that predictive learning induces a persistent accumulation of delta-opioid receptors (DOPrs) at the somatic membrane of cholinergic interneurons (CINs) in the nucleus accumbens shell (Nac-S). This accumulation is required for predictive learning to influence subsequent choice between goal-directed actions. The current experiments investigated the local neurochemical events responsible for this translocation. We found that (1) local administration of substance P into multiple striatal sub-territories induced DOPr translocation and (2) that this effect was mediated by the NK1 receptor, likely through its expression on CINs. Interestingly, whereas intrastriatal infusion of the D1 agonist chloro-APB reduced the DOPr translocation on CINs and infusion of the D2 agonist quinpirole had no effect, co-administration of both agonists again generated DOPr translocation, suggesting the effect of the D1 agonist alone was due to receptor internalisation. In support of this, local administration of cocaine was found to increase DOPr translocation as was chloro-APB when co-administered with the DOPr antagonist naltrindole. These studies provide the first evidence of delta-opioid receptor translocation in striatal cholinergic interneurons outside of the accumbens shell and suggest that, despite differences in local striatal neurochemical microenvironments, a similar molecular mechanism - involving an interaction between dopamine and SP signalling via NK1R - regulates DOPr translocation in multiple striatal regions. To our knowledge, this represents a novel mechanism by which DOPr distribution is regulated that may be particularly relevant to learning-induced DOPr trafficking.

Chronic Morphine Reduces Surface Expression of δ-Opioid Receptors in Subregions of Rostral Striatum.

The delta opioid receptor (DOPr), whilst not the primary target of clinically used opioids, is involved in development of opioid tolerance and addiction. There is growing evidence that DOPr trafficking is involved in drug addiction, e.g., a range of studies have shown increased plasma membrane DOPr insertion during chronic treatment with opioids. The present study used a transgenic mouse model in which the C-terminal of the DOPr is tagged with enhanced-green fluorescence protein to examine the effects of chronic morphine treatment on surface membrane expression in striatal cholinergic interneurons that are implicated in motivated learning following both chronic morphine and morphine sensitization treatment schedules in male mice. A sex difference was noted throughout the anterior striatum, which was most prominent in the nucleus accumbens core region. Incontrast with previous studies in other neurons, chronic exposure to a high dose of morphine for 6 days had no effect, or slightly decreased (anterior dorsolateral striatum) surface DOPr expression. A morphine sensitization schedule produced similar results with a significant decrease in surface DOPr expression in nucleus accumbens shell. These results suggest that chronic morphine and morphine sensitisation treatment may have effects on instrumental reward-seeking behaviours and learning processes related to drug addiction, via effects on striatal DOPr function.

Inhibition of the norepinephrine transporter by χ-conotoxin dendrimers.

Peptide dendrimers are a novel class of macromolecules of emerging interest with the potential of delayed renal clearance due to their molecular size and enhanced activity due to the multivalency effect. In this work, an active analogue of the disulfide-rich χ-conotoxin χ-MrIA (χ-MrIA), a norepinephrine reuptake (norepinephrine transporter) inhibitor, was grafted onto a polylysine dendron. Dendron decoration was achieved by employing copper-catalyzed alkyne-azide cycloaddition with azido-PEG chain-modified χ-MrIA analogues, leading to homogenous 4-mer and 8-mer χ-MrIA dendrimers with molecular weights ranging from 8 to 22 kDa. These dendrimers were investigated for their impact on peptide secondary structure, in vitro functional activity, and potential anti-allodynia in vivo. NMR studies showed that the χ-MrIA tertiary structure was maintained in the χ-MrIA dendrimers. In a functional norepinephrine transporter reuptake assay, χ-MrIA dendrimers showed slightly increased potency relative to the azido-PEGylated χ-MrIA analogues with similar potency to the parent peptide. In contrast to χ-MrIA, no anti-allodynic action was observed when the χ-MrIA dendrimers were administered intrathecally in a rat model of neuropathic pain, suggesting that the larger dendrimer structures are unable to diffuse through the spinal column tissue and reach the norepinephrine transporter. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Regulation of µ-opioid receptors: desensitization, phosphorylation, internalization, and tolerance.

Morphine and related µ-opioid receptor (MOR) agonists remain among the most effective drugs known for acute relief of severe pain. A major problem in treating painful conditions is that tolerance limits the long-term utility of opioid agonists. Considerable effort has been expended on developing an understanding of the molecular and cellular processes that underlie acute MOR signaling, short-term receptor regulation, and the progression of events that lead to tolerance for different MOR agonists. Although great progress has been made in the past decade, many points of contention and controversy cloud the realization of this progress. This review attempts to clarify some confusion by clearly defining terms, such as desensitization and tolerance, and addressing optimal pharmacological analyses for discerning relative importance of these cellular mechanisms. Cellular and molecular mechanisms regulating MOR function by phosphorylation relative to receptor desensitization and endocytosis are comprehensively reviewed, with an emphasis on agonist-biased regulation and areas where knowledge is lacking or controversial. The implications of these mechanisms for understanding the substantial contribution of MOR signaling to opioid tolerance are then considered in detail. While some functional MOR regulatory mechanisms contributing to tolerance are clearly understood, there are large gaps in understanding the molecular processes responsible for loss of MOR function after chronic exposure to opioids. Further elucidation of the cellular mechanisms that are regulated by opioids will be necessary for the successful development of MOR-based approaches to new pain therapeutics that limit the development of tolerance.

The acquisition of goal-directed actions generates opposing plasticity in direct and indirect pathways in dorsomedial striatum.

A cortical-basal ganglia network involving, particularly, the posterior region of dorsomedial striatum (DMS) has been implicated in the acquisition of goal-directed actions; however, no direct evidence of learning-related plasticity in this striatal region has been reported, nor is it known whether, or which, specific cell types are involved in this learning process. The striatum is primarily composed of two classes of spiny projection neurons (SPNs): the striatonigral and striatopallidal SPNs, which express dopamine D1 and D2 receptors, respectively. Here we establish that, in mice, the acquisition of goal-directed actions induced plasticity in both D1- and D2-SPNs specifically in the DMS and, importantly, that these changes were in opposing directions; after learning, AMPA/NMDA ratios were increased in D1-SPNs and reduced in the D2-SPNs in the DMS. Such opposing plasticity could provide the basis for rapidly rebiasing the control of task-specific actions, and its dysregulation could underlie disorders associated with striatal function.

A Positive Allosteric Modulator of the Adenosine A1 Receptor Selectively Inhibits Primary Afferent Synaptic Transmission in a Neuropathic Pain Model.

In the spinal cord and periphery, adenosine inhibits neuronal activity through activation of the adenosine A1 receptor (A1R), resulting in antinociception and highlighting the potential of therapeutically targeting the receptor in the treatment of neuropathic pain. This study investigated the changes in adenosine tone and A1R signaling, together with the actions of a novel A1R positive allosteric modulator (PAM), VCP171 [(2-amino-4-(3-(trifluoromethyl)phenyl)thiophen-3-yl)(phenyl)methanone], on excitatory and inhibitory neurotransmission at spinal cord superficial dorsal horn synapses in a rat partial nerve-injury model of neuropathic pain. In the absence of A1R agonists, superfusion of the A1R antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 1 µM), produced a significantly greater increase in electrically evoked α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated synaptic current (eEPSC) amplitude in both lamina I and II neurons from nerve-injured animals than in controls, suggesting that endogenous adenosine tone is increased in the dorsal horn. Inhibitory GABAergic and glycinergic synaptic currents were also significantly increased by DPCPX in controls but there was no difference after nerve injury. The A1R agonist, N6-cyclopentyladenosine, produced greater inhibition of eEPSC amplitude in lamina II but not lamina I of the spinal cord dorsal horn in nerve-injured versus control animals, suggesting a functional increase in A1R sensitivity in lamina II neurons after nerve injury. The A1R PAM, VCP171, produced a greater inhibition of eEPSC amplitude of nerve-injury versus control animals in both lamina I and lamina II neurons. Enhanced adenosine tone and A1R sensitivity at excitatory synapses in the dorsal horn after nerve injury suggest that new generation PAMs of the A1R can be effective treatments for neuropathic pain.

Role of Phosphorylation Sites in Desensitization of µ-Opioid Receptor.

Phosphorylation of residues in the C-terminal tail of the µ-opioid receptor (MOPr) is thought to be a key step in desensitization and internalization. Phosphorylation of C-terminal S/T residues is required for internalization (Just et al., 2013), but its role in desensitization is unknown. This study examined the influence of C-terminal phosphorylation sites on rapid desensitization of MOPr. Wild-type MOPr, a 3S/T-A mutant (S363A, T370A, S375A) that maintains internalization, 6S/T-A (S363A, T370A, S375A, T376A, T379A, T383A) and 11S/T-A (all C-terminal S/T residues mutated) mutants not internalized by MOPr agonists were stably expressed in AtT20 cells. Perforated patch-clamp recordings of MOPr-mediated activation of G-protein-activated inwardly rectifying potassium channel (Kir3.X) (GIRK) conductance by submaximal concentrations of Met(5)-enkephalin (ME) and somatostatin (SST; coupling to native SST receptor [SSTR]) were used to examine desensitization induced by exposure to ME and morphine for 5 minutes at 37°C. The rates of ME- and morphine-induced desensitization did not correlate with phosphorylation using phosphorylation site-specific antibodies. ME-induced MOPr desensitization and resensitization did not differ from wild-type for 3S/T-A and 6S/T-A but was abolished in 11S/T-A. Morphine-induced desensitization was unaffected in all three mutants, as was heterologous desensitization of SSTR. Morphine-induced desensitization (but not ME) was reduced by protein kinase C inhibition in wild-type MOPr and abolished in the 11S/T-A mutant, as was heterologous desensitization. These findings establish that MOPr desensitization can occur independently of S/T phosphorylation and internalization; however, C-terminal phosphorylation is necessary for some forms of desensitization because mutation of all C-terminal sites (11S/T-A) abolishes desensitization induced by ME.

High-voltage-activated calcium current subtypes in mouse DRG neurons adapt in a subpopulation-specific manner after nerve injury.

Changes in ion channel function and expression are characteristic of neuropathic pain. Voltage-gated calcium channels (VGCCs) are integral for neurotransmission and membrane excitability, but relatively little is known about changes in their expression after nerve injury. In this study, we investigate whether peripheral nerve ligation is followed by changes in the density and proportion of high-voltage-activated (HVA) VGCC current subtypes in dorsal root ganglion (DRG) neurons, the contribution of presynaptic N-type calcium channels in evoked excitatory postsynaptic currents (EPSCs) recorded from dorsal horn neurons in the spinal cord, and the changes in expression of mRNA encoding VGCC subunits in DRG neurons. Using C57BL/6 mice [8- to 11-wk-old males (n = 91)] for partial sciatic nerve ligation or sham surgery, we performed whole cell patch-clamp recordings on isolated DRG neurons and dorsal horn neurons and measured the expression of all VGCC subunits with RT-PCR in DRG neurons. After nerve injury, the density of P/Q-type current was reduced overall in DRG neurons. There was an increase in the percentage of N-type and a decrease in that of P/Q-type current in medium- to large-diameter neurons. No changes were found in the contribution of presynaptic N-type calcium channels in evoked EPSCs recorded from dorsal horn neurons. The α2δ-1 subunit was upregulated by 1.7-fold and γ-3, γ-2, and ß-4 subunits were all downregulated 1.7-fold in injured neurons compared with sham-operated neurons. This comprehensive characterization of HVA VGCC subtypes in mouse DRG neurons after nerve injury revealed changes in N- and P/Q-type current proportions only in medium- to large-diameter neurons.

Plasticity in striatopallidal projection neurons mediates the acquisition of habitual actions.

In instrumental conditioning, newly acquired actions are generally goal-directed and are mediated by the relationship between the action and its consequences or outcome. With continued training, however, the performance of such actions can become automatic, reflexive or habitual and under the control of antecedent stimuli rather than their consequences. Recent evidence suggests that habit learning is mediated by plasticity in the dorsolateral striatum (DLS). To date, however, no direct evidence of learning-related plasticity associated with overtraining has been reported in this region, nor is it known whether, or which, specific cell types are involved in this learning process. The striatum is primarily composed of two classes of spiny projection neurons, the striatonigral and striatopallidal spiny projection neurons, which express dopamine D1 and D2 receptors, and control direct and indirect pathways, respectively. Here we found evidence of a post-synaptic depression in DLS striatopallidal projecting neurons in the indirect pathway during habit learning in mice. Moreover, this training-induced depression occluded post-synaptic depression induced by co-activation of D2 receptors and transient receptor potential vanilloid 1 (TRPV1) channels, implying that this pathway is involved in habit learning. This hypothesis was further tested by disrupting this signal pathway by knocking out TRPV1 channels, resulting in compromised habit learning. Our findings suggest that post-synaptic plasticity at D2 neurons in the DLS mediates habit learning and, by implicating an interaction between the D2 receptor and TRPV1 channel activity, provide a potential drug target for influencing habitual action control.

Conus venom peptide pharmacology.

Conopeptides are a diverse group of recently evolved venom peptides used for prey capture and/or defense. Each species of cone snails produces in excess of 1000 conopeptides, with those pharmacologically characterized (≈ 0.1%) targeting a diverse range of membrane proteins typically with high potency and specificity. The majority of conopeptides inhibit voltage- or ligand-gated ion channels, providing valuable research tools for the dissection of the role played by specific ion channels in excitable cells. It is noteworthy that many of these targets are found to be expressed in pain pathways, with several conopeptides having entered the clinic as potential treatments for pain [e.g., pyroglutamate1-MrIA (Xen2174)] and one now marketed for intrathecal treatment of severe pain [ziconotide (Prialt)]. This review discusses the diversity, pharmacology, structure-activity relationships, and therapeutic potential of cone snail venom peptide families acting at voltage-gated ion channels (ω-, µ-, µO-, δ-, ι-, and κ-conotoxins), ligand-gated ion channels (α-conotoxins, σ-conotoxin, ikot-ikot, and conantokins), G-protein-coupled receptors (ρ-conopeptides, conopressins, and contulakins), and neurotransmitter transporters (χ-conopeptides), with expanded discussion on the clinical potential of sodium and calcium channel inhibitors and α-conotoxins. Expanding the discovery of new bioactives using proteomic/transcriptomic approaches combined with high-throughput platforms and better defining conopeptide structure-activity relationships using relevant membrane protein crystal structures are expected to grow the already significant impact conopeptides have had as both research probes and leads to new therapies.

Stabilization of the cysteine-rich conotoxin MrIA by using a 1,2,3-triazole as a disulfide bond mimetic.

The design of disulfide bond mimetics is an important strategy for optimising cysteine-rich peptides in drug development. Mimetics of the drug lead conotoxin MrIA, in which one disulfide bond is selectively replaced of by a 1,4-disubstituted-1,2,3-triazole bridge, are described. Sequential copper-catalyzed azide-alkyne cycloaddition (CuAAC; click reaction) followed by disulfide formation resulted in the regioselective syntheses of triazole-disulfide hybrid MrIA analogues. Mimetics with a triazole replacing the Cys4-Cys13 disulfide bond retained tertiary structure and full in vitro and in vivo activity as norepinephrine reuptake inhibitors. Importantly, these mimetics are resistant to reduction in the presence of glutathione, thus resulting in improved plasma stability and increased suitability for drug development.