Time-Dependent Changes in Protein Composition of Medial Prefrontal Cortex in Rats with Neuropathic Pain.

Chronic pain is associated with time-dependent structural and functional reorganization of the prefrontal cortex that may reflect adaptive pain compensatory and/or maladaptive pain-promoting mechanisms. However, the molecular underpinnings of these changes and whether there are time-dependent relationships to pain progression are not well characterized. In this study, we analyzed protein composition in the medial prefrontal cortex (mPFC) of rats at two timepoints after spinal nerve ligation (SNL) using two-dimensional gel electrophoresis (2D-ELFO) and liquid chromatography with tandem mass spectrometry (LC-MS/MS). SNL, but not sham-operated, rats developed persistent tactile allodynia and thermal hyperalgesia, confirming the presence of experimental neuropathic pain. Two weeks after SNL (early timepoint), we identified 11 proteins involved in signal transduction, protein transport, cell homeostasis, metabolism, and apoptosis, as well as heat-shock proteins and chaperones that were upregulated by more than 1.5-fold compared to the sham-operated rats. Interestingly, there were only four significantly altered proteins identified at 8 weeks after SNL (late timepoint). These findings demonstrate extensive time-dependent modifications of protein expression in the rat mPFC under a chronic neuropathic pain state that might underlie the evolution of chronic pain characterized by early pain-compensatory and later aberrant mechanisms.

Preclinical Assessment of the Analgesic Pharmacology of NKTR-181 in Rodents.

OBJECTIVE: Pharmacological evaluation of the mu-opioid receptor (MOR) agonist properties of NKTR-181 in rodent models. METHODS: Graded noxious stimulus intensities were used in rats to establish the antinociceptive potency and efficacy of NKTR-181 relative to morphine, fentanyl, and oxycodone. Characteristics of MOR agonist actions, as measured by antinociceptive tolerance and cross-tolerance, as well as opioid-induced hyperalgesia (OIH) and naloxone-precipitated withdrawal in NKTR-181- and morphine-dependent in mice, were compared. RESULTS: NKTR-181 showed dose- and time-related antinociception with similar maximal effects to morphine in the rat and mouse hot-water tail-flick test. No sex or species differences were observed in NKTR-181 or morphine antinociception. Rats treated with NKTR-181 and morphine exhibited decreases in both potency and maximal efficacy as nociceptive stimulus intensity was increased from a water temperature of 50 °C to 54 °C. Evaluation of antinociception at a high stimulus intensity revealed that oxycodone and fentanyl exhibited greater efficacy than either NKTR-181 or morphine. The relative potency difference between NKTR-181 and morphine across all tail-flick studies was determined to be 7.6-fold (90% confidence interval, 2.6, 21.5). The peak antinociceptive effect of NKTR-181 was delayed compared to that of the other opioids and cumulative drug effects were not observed. Repeated treatment with escalating, approximately equi-analgesic doses of NKTR-181 or morphine, produced antinociceptive tolerance and cross-tolerance. Under these pharmacological conditions, OIH and naloxone-precipitated physical dependence were similar for NKTR-181 and morphine. CONCLUSIONS: NKTR-181 had a slower onset, but similar efficacy, to morphine in the models studied supporting reduced abuse potential while maintaining analgesic effect in comparison with current opioids.

C-terminal modified Enkephalin-like tetrapeptides with enhanced affinities at the kappa opioid receptor and monoamine transporters.

A new series of enkephalin-like tetrapeptide analogs modified at the C-terminus by an N-(3,4-dichlorophenyl)-N-(piperidin-4-yl)propionamide (DPP) moiety were designed, synthesized, and tested for their binding affinities at opioid receptors and monoamine transporters to evaluate their potential multifunctional activity for the treatment of chronic pain. Most ligands exhibited high binding affinities in the nanomolar range at the opioid receptors with a slight delta-opioid receptor (DOR) selectivity over mu-opioid receptor (MOR) and kappa-opioid receptor (KOR) and low binding affinities in the micromolar range at the monoamine transporters, SERT and NET. Ligands of which the positions 1 and 4 were substituted by Dmt and Phe(4-X) residues, respectively, showed the excellent binding affinities at three opioid receptors. Among them, Dmt-d-Tic-Gly-Phe(4-F)-DPP was the most promising considering its excellent opioid affinities, particularly unexpected high binding affinity (Ki = 0.13 nM) at the KOR, and moderate interactions with serotonin/norepinephrine reuptake inhibitors (SNRIs). Docking studies revealed that the ligand was a good fit for the KOR binding pocket (binding score = 8,750).

Cyclic biphalin analogues with a novel linker lead to potent agonist activities at mu, delta, and kappa opioid receptors.

In an effort to improve biphalin's potency and efficacy at the µ-(MOR) and δ-opioid receptors (DOR), a series of cyclic biphalin analogues 1-5 with a cystamine or piperazine linker at the C-terminus were designed and synthesized by solution phase synthesis using Boc-chemistry. Interestingly, all of the analogues showed balanced opioid agonist activities at all opioid receptor subtypes due to enhanced κ-opioid receptor (KOR) activity. Our results indicate that C-terminal flexible linkers play an important role in KOR activity compared to that of the other cyclic biphalin analogues with a hydrazine linker. Among them, analogue 5 is a potent (Ki = 0.27, 0.46, and 0.87 nM; EC50 = 3.47, 1.45, and 13.5 nM at MOR, DOR, and KOR, respectively) opioid agonist with high efficacy. Based on the high potency and efficacy at the three opioid receptor subtypes, the ligand is expected to have a potential synergistic effect on relieving pain and further studies including in vivo tests are worthwhile.

Cyclic non-opioid dynorphin A analogues for the bradykinin receptors.

Nerve injury and inflammation cause up-regulation of an endogenous opioid ligand, dynorphin A (Dyn A), in the spinal cord resulting in hyperalgesia via the interaction with bradykinin receptors (BRs). This is a non-opioid neuroexcitatory effect that cannot be blocked by opioid antagonists. Our systematic structure-activity relationships study on Dyn A identified lead ligands 1 and 4, along with the key structural feature (i.e. amphipathicity) for the BRs. However, the ligands showed very low metabolic stability in plasma (t1/2 <1h) and therefore, in order to improve their metabolic stabilities with retained biological activities, various modifications were performed. Cyclization of ligand 4 afforded a cyclic Dyn A analogue 5 that retained the same range of binding affinity as the linear ligand with improved metabolic stability (t1/2 >5h) and therefore possesses the potential as a pharmacophoric scaffold to be utilized for drug development.

Modification of amphipathic non-opioid dynorphin A analogues for rat brain bradykinin receptors.

It has been shown that under chronic pain or nerve injury conditions, up-regulated dynorphin A (Dyn A) interacts with bradykinin receptors (BRs) to cause hyperalgesia in the spinal cord. Thus BRs antagonist can modulate hyperalgesia by blocking Dyn A's interaction with the BRs in the central nervous system. In our earlier structure-activity relationship (SAR) study, [des-Arg(7)]-Dyn A-(4-11) 13 was discovered as a minimum pharmacophore for rat brain BRs with its antagonist activity (anti-hyperalgesic effect) in in vivo tests using naïve or injured animals. We have pursued further modification on the [des-Arg(7)]-Dyn A analogues and identified a key insight into the pharmacophore of the rat brain BRs: amphipathicity.

Design, synthesis, and biological evaluation of a series of bifunctional ligands of opioids/SSRIs.

A series of opioid and serotonin re-uptake inhibitors (SSRIs) bifunctional ligands have been designed, synthesized, and tested for their activities and efficacies at µ-, δ- and κ opioid receptors and SSRIs receptors. Most of the compounds showed high affinities for µ- and δ-opioid receptors and lower affinities for SSRIs and κ opioid receptors. A docking study on the µ-opioid receptor binding pocket has been carried out for ligands 3-11. The ligands 7 and 11 have displayed the highest binding profiles for the µ-opioid receptor binding site with ΔGbind (-12.14kcal/mol) and Ki value (1.0nM), and ΔGbind (-12.41kcal/mol) and Ki value (0.4nM), respectively. Ligand 3 was shown to have the potential of dual acting serotonin/norepinephrine re-uptake inhibitor (SNRI) antidepressant activity in addition to opioid activities, and thus could be used for the design of multifunctional ligands in the area of a novel approach for the treatment of pain and depression.

Discovery of amphipathic dynorphin A analogues to inhibit the neuroexcitatory effects of dynorphin A through bradykinin receptors in the spinal cord.

We hypothesized that under chronic pain conditions, up-regulated dynorphin A (Dyn A) interacts with bradykinin receptors (BRs) in the spinal cord to promote hyperalgesia through an excitatory effect, which is opposite to the well-known inhibitory effect of opioid receptors. Considering the structural dissimilarity between Dyn A and endogenous BR ligands, bradykinin (BK) and kallidin (KD), this interaction could not be predicted, but it allowed us to discover a potential neuroexcitatory target. Well-known BR ligands, BK, [des-Arg(10), Leu(9)]-kallidin (DALKD), and HOE140 showed different binding profiles at rat brain BRs than that previously reported. These results suggest that neuronal BRs in the rat central nervous system (CNS) may be pharmacologically distinct from those previously defined in non-neuronal tissues. Systematic structure-activity relationship (SAR) study at the rat brain BRs was performed, and as a result, a new key structural feature of Dyn A for BR recognition was identified: amphipathicity. NMR studies of two lead ligands, Dyn A-(4-11) 7 and [des-Arg(7)]-Dyn A-(4-11) 14, which showed the same high binding affinity, confirmed that the Arg residue in position 7, which is known to be crucial for Dyn A's biological activity, is not necessary, and that a type I ß-turn structure at the C-terminal part of both ligands plays an important role in retaining good binding affinities at the BRs. Our lead ligand 14 blocked Dyn A-(2-13) 10-induced hyperalgesic effects and motor impairment in in vivo assays using naïve rats. In a model of peripheral neuropathy, intrathecal (i.th.) administration of ligand 14 reversed thermal hyperalgesia and mechanical hypersensitivity in a dose-dependent manner in nerve-injured rats. Thus, ligand 14 may inhibit abnormal pain states by blocking the neuroexcitatory effects of enhanced levels of Dyn A, which are likely to be mediated by BRs in the spinal cord.

Structure-activity relationships of non-opioid [des-Arg(7)]-dynorphin A analogues for bradykinin receptors.

In our earlier studies, bradykinin receptors (BRs) were identified as a potential target for the neuroexcitatory effects of dynorphin A (Dyn A) in the central nervous system (CNS), and [des-Arg(7)]-Dyn A-(4-11) (6) was discovered as a lead ligand to modulate Dyn A-(2-13) induced neuroexcitatory effects in the CNS as an antagonist. In an effort to gain insights into key structural features of the Dyn A for the BRs, we pursued further structure-activity relationships (SAR) study on the [des-Arg(7)]-Dyn A analogs and confirmed that all of the [des-Arg(7)]-Dyn A analogues showed good binding affinities at the BRs.

Synthesis and evaluation of bivalent ligands for binding to the human melanocortin-4 receptor.

Membrane proteins, especially G-protein coupled receptors (GPCRs), are interesting and important theragnostic targets since many of them serve in intracellular signaling critical for all aspects of health and disease. The potential utility of designed bivalent ligands as targeting agents for cancer diagnosis and/or therapy can be evaluated by determining their binding to the corresponding receptors. As proof of concept, GPCR cell surface proteins are shown to be targeted specifically using multivalent ligands. We designed, synthesized, and tested a series of bivalent ligands targeting the over-expressed human melanocortin 4 receptor (hMC4R) in human embryonic kidney (HEK) 293 cells. Based on our data suggesting an optimal linker length of 25±10Å inferred from the bivalent melanocyte stimulating hormone (MSH) agonist, the truncated heptapeptide, referred to as MSH(7): Ac-Ser-Nle-Glu-His-D-Phe-Arg-Trp-NH2 was used to construct a set of bivalent ligands incorporating a hMC4R antagonist, SHU9119: Ac-Nle-c[Asp-His-2'-D-Nal-Arg-Trp-Lys]-NH2 and another set of bivalent ligands containing the SHU9119 antagonist pharmacophore on both side of the optimized linkers. These two binding motifs within the bivalent constructs were conjoined by semi-rigid (Pro-Gly)3 units with or without the flexible poly(ethylene glycol) (PEGO) moieties. Lanthanide-based competitive binding assays showed bivalent ligands binds to the hMC4R with up to 240-fold higher affinity than the corresponding linked monovalent ligands.

The impact of multifunctional enkephalin analogs and morphine on the protein changes in crude membrane fractions isolated from the rat brain cortex and hippocampus.

Endogenous opioid peptides serve as potent analgesics through the opioid receptor (OR) activation. However, they often suffer from poor metabolic stability, low lipophilicity, and low blood-brain barrier permeability. Researchers have developed many strategies to overcome the drawbacks of current pain medications and unwanted biological effects produced by the interaction with opioid receptors. Here, we tested multifunctional enkephalin analogs LYS739 (MOR/DOR agonist and KOR partial antagonist) and LYS744 (MOR/DOR agonist and KOR full antagonist) under in vivo conditions in comparison with MOR agonist, morphine. We applied 2D electrophoretic resolution to investigate differences in proteome profiles of crude membrane (CM) fractions isolated from the rat brain cortex and hippocampus exposed to the drugs (10 mg/kg, seven days). Our results have shown that treatment with analog LYS739 induced the most protein changes in cortical and hippocampal samples. The identified proteins were mainly associated with energy metabolism, cell shape and movement, apoptosis, protein folding, regulation of redox homeostasis, and signal transduction. Among these, the isoform of mitochondrial ATP synthase subunit beta (ATP5F1B) was the only protein upregulation in the hippocampus but not in the brain cortex. Contrarily, the administration of analog LYS744 caused a small number of protein alterations in both brain parts. Our results indicate that the KOR full antagonism, together with MOR/DOR agonism of multifunctional opioid ligands, can be beneficial in treating chronic pain states by reducing changes in protein expression levels but retaining analgesic efficacy.

Effect of anchoring 4-anilidopiperidines to opioid peptides.

We report here the design, synthesis, and in vitro characterization of new opioid peptides featuring a 4-anilidopiperidine moiety. Despite the fact that the chemical structures of fentanyl surrogates have been found suboptimal per se for the opioid activity, the corresponding conjugates with opioid peptides displayed potent opioid activity. These studies shed an instructive light on the strategies and potential therapeutic values of anchoring the 4-anilidopiperidine scaffold to different classes of opioid peptides.

Peptidomimetics and Their Applications for Opioid Peptide Drug Discovery.

Despite various advantages, opioid peptides have been limited in their therapeutic uses due to the main drawbacks in metabolic stability, blood-brain barrier permeability, and bioavailability. Therefore, extensive studies have focused on overcoming the problems and optimizing the therapeutic potential. Currently, numerous peptide-based drugs are being marketed thanks to new synthetic strategies for optimizing metabolism and alternative routes of administration. This tutorial review briefly introduces the history and role of natural opioid peptides and highlights the key findings on their structure-activity relationships for the opioid receptors. It discusses details on opioid peptidomimetics applied to develop therapeutic candidates for the treatment of pain from the pharmacological and structural points of view. The main focus is the current status of various mimetic tools and the successful applications summarized in tables and figures.

The Dose-Dependent Effects of Multifunctional Enkephalin Analogs on the Protein Composition of Rat Spleen Lymphocytes, Cortex, and Hippocampus; Comparison with Changes Induced by Morphine.

This work aimed to test the effect of 7-day exposure of rats to multifunctional enkephalin analogs LYS739 and LYS744 at doses of 3 mg/kg and 10 mg/kg on the protein composition of rat spleen lymphocytes, brain cortex, and hippocampus. Alterations of proteome induced by LYS739 and LYS744 were compared with those elicited by morphine. The changes in rat proteome profiles were analyzed by label-free quantification (MaxLFQ). Proteomic analysis indicated that the treatment with 3 mg/kg of LYS744 caused significant alterations in protein expression levels in spleen lymphocytes (45), rat brain cortex (31), and hippocampus (42). The identified proteins were primarily involved in RNA processing and the regulation of cytoskeletal dynamics. In spleen lymphocytes, the administration of the higher 10 mg/kg dose of both enkephalin analogs caused major, extensive modifications in protein expression levels: LYS739 (119) and LYS744 (182). Among these changes, the number of proteins associated with immune responses and apoptotic processes was increased. LYS739 treatment resulted in the highest number of alterations in the rat brain cortex (152) and hippocampus (45). The altered proteins were functionally related to the regulation of transcription and cytoskeletal reorganization, which plays an essential role in neuronal plasticity. Administration with LYS744 did not increase the number of altered proteins in the brain cortex (26) and hippocampus (26). Our findings demonstrate that the effect of κ-OR full antagonism of LYS744 is opposite in the central nervous system and the peripheral region (spleen lymphocytes).

Regulation of mitochondrial fission by GIPC-mediated Drp1 retrograde transport.

Dynamin-related protein 1 (Drp1) is a key regulator of mitochondrial fission, a large cytoplasmic GTPase recruited to the mitochondrial surface via transmembrane adaptors to initiate scission. While Brownian motion likely accounts for the local interactions between Drp1 and the mitochondrial adaptors, how this essential enzyme is targeted from more distal regions like the cell periphery remains unknown. Based on proteomic interactome screening and cell-based studies, we report that GAIP/RGS19-interacting protein (GIPC) mediates the actin-based retrograde transport of Drp1 toward the perinuclear mitochondria to enhance fission. Drp1 interacts with GIPC through its atypical C-terminal PDZ-binding motif. Loss of this interaction abrogates Drp1 retrograde transport resulting in cytoplasmic mislocalization and reduced fission despite retaining normal intrinsic GTPase activity. Functionally, we demonstrate that GIPC potentiates the Drp1-driven proliferative and migratory capacity in cancer cells. Together, these findings establish a direct molecular link between altered GIPC expression and Drp1 function in cancer progression and metabolic disorders.

ßIV-spectrin as a stalk cell-intrinsic regulator of VEGF signaling.

Defective angiogenesis underlies over 50 malignant, ischemic and inflammatory disorders yet long-term therapeutic applications inevitably fail, thus highlighting the need for greater understanding of the vast crosstalk and compensatory mechanisms. Based on proteomic profiling of angiogenic endothelial components, here we report ßIV-spectrin, a non-erythrocytic cytoskeletal protein, as a critical regulator of sprouting angiogenesis. Early loss of endothelial-specific ßIV-spectrin promotes embryonic lethality in mice due to hypervascularization and hemorrhagic defects whereas neonatal depletion yields higher vascular density and tip cell populations in developing retina. During sprouting, ßIV-spectrin expresses in stalk cells to inhibit their tip cell potential by enhancing VEGFR2 turnover in a manner independent of most cell-fate determining mechanisms. Rather, ßIV-spectrin recruits CaMKII to the plasma membrane to directly phosphorylate VEGFR2 at Ser984, a previously undefined phosphoregulatory site that strongly induces VEGFR2 internalization and degradation. These findings support a distinct spectrin-based mechanism of tip-stalk cell specification during vascular development.

Multifunctional Enkephalin Analogs with a New Biological Profile: MOR/DOR Agonism and KOR Antagonism.

In our previous studies, we developed a series of mixed MOR/DOR agonists that are enkephalin-like tetrapeptide analogs with an N-phenyl-N-piperidin-4-ylpropionamide (Ppp) moiety at the C-terminus. Further SAR study on the analogs, initiated by the findings from off-target screening, resulted in the discovery of LYS744 (6, Dmt-DNle-Gly-Phe(p-Cl)-Ppp), a multifunctional ligand with MOR/DOR agonist and KOR antagonist activity (GTPγS assay: IC50 = 52 nM, Imax = 122% cf. IC50 = 59 nM, Imax = 100% for naloxone) with nanomolar range of binding affinity (Ki = 1.3 nM cf. Ki = 2.4 nM for salvinorin A). Based on its unique biological profile, 6 is considered to possess high therapeutic potential for the treatment of chronic pain by modulating pathological KOR activation while retaining analgesic efficacy attributed to its MOR/DOR agonist activity.

Design and synthesis of trivalent ligands targeting opioid, cholecystokinin, and melanocortin receptors for the treatment of pain.

It has been known that co-administration of morphine with either cholecystokinin (CCK) receptor or melanocortin (MC) receptor antagonists enhance morphine's analgesic efficacy by reducing serious side effects such as tolerance and addiction. Considering these synergistic effects, we have designed trivalent ligands in which all three different pharmacophores for opioid, CCK, and MC receptors are combined in such a way as to conserve their own topographical pharmacophore structures. These ligands, excluding the cyclic compound, were synthesized by solid phase synthesis using Rink-amide resin under microwave assistance in very high yields. These trivalent ligands bind to their respective receptors well demonstrating that the topographical pharmacophore structures for the three receptors were retained for receptor binding. Ligand 10 was a lead compound to show the best biological activities at all three receptors.

Effectiveness and safety of electrical moxibustion for knee osteoarthritis: A multicenter, randomized, assessor-blinded, parallel-group clinical trial.

BACKGROUND: The prevalence of knee osteoarthritis (KOA) is increasing, and it has emerged as a major health issue. Studies have been reported that moxibustion is effective for treating KOA, but conventional moxibustion is difficult to control the intensity of stimulation and causes smoke, harmful gases, or odors. An electrical moxibustion (EM) device was developed to solve these problems, so we conducted this study to evaluate the effectiveness and safety of EM as a treatment for KOA. METHODS: This is a multicenter, randomized, assessor-blinded, parallel-group clinical trial. Participants with KOA were randomly allocated into EM, traditional indirect moxibustion (TIM), or usual care groups. The moxibustion groups were received 12 sessions of moxibustion treatment at six acupuncture points (ST36, ST35, ST34, SP9, EX-LE4, SP10) over a period of 6 weeks. The usual care group was received usual treatment and self-care. The primary outcome was the degree of pain measured by numerical rating scale (NRS). The second outcomes were measured using visual analog scale, Korean version of the Western Ontario and McMaster Universities osteoarthritis index, patient global assessment, European quality of life five dimension five level scale, and warm sense threshold and heat pain threshold. For safety assessment, laboratory test and adverse events (AEs) were recorded. RESULTS: A total of 138 participants were assigned. While there was no significant NRS change in the usual care, EM and TIM showed significant decrease after treatment. Compared to the usual care, the mean change of NRS in the EM and TIM was significantly different, but there was no significance between two groups. Regarding secondary outcomes, EM and TIM also showed significant difference compared to the usual care, but there was no significance between two groups. Regarding safety assessment, while usual care showed significant safety among three groups, EM showed seven treatment-related AEs by four participants compared TIM's 10 events by 10 participants. In addition, there was no blister caused by burns in the EM, which occurred four cases in the TIM. CONCLUSION: This study shows that EM is effective to improve the pain and function by KOA with a certain level of safety.

Gram-Scale Preparation of C-Terminal-Modified Enkephalin Analogues by Typical Liquid-Phase Peptide Synthesis.

This article describes the gram-scale liquid-phase peptide synthesis of C-terminal-modified enkephalin analogues that possess high analgesic efficacy in animals, high potency for mu and delta opioid receptors, and high metabolic stability and potential blood-brain barrier permeability. Despite the long cycle time and tedious purification steps, liquid-phase synthesis is still a preferred method for large-scale peptide synthesis due to its cost effectiveness (i.e., amount of amino acids and reagents required), easy detection, and isolation of impurities compared with solid-phase synthesis. A robust liquid-phase synthesis protocol is described, involving BOP-assisted coupling and Boc deprotection, which has been well established in the laboratory and is a useful synthetic protocol for cost-effective production of peptide drugs. © 2019 by John Wiley & Sons, Inc.