Applications of biaryl cyclization in the synthesis of cyclic enkephalin analogs with a highly restricted flexibility.

A series of 10 cyclic, biaryl analogs of enkephalin, with Tyr or Phe residues at positions 1 and 4, were synthesized according to the Miyaura borylation and Suzuki coupling methodology. Biaryl bridges formed by side chains of the two aromatic amino acid residues are of the meta-meta, meta-para, para-meta, and para-para configuration. Conformational properties of the peptides were studied by CD and NMR. CD studies allowed only to compare conformations of individual peptides while NMR investigations followed by XPLOR calculations provided detailed information on their conformation. Reliability of the XPLOR calculations was confirmed by quantum chemical ones performed for one of the analogs. No intramolecular hydrogen bonds were found in all the peptides. They are folded and adopt the type IV ß-turn conformation. Due to a large steric strain, the aromatic carbon atoms forming the biaryl bond are distinctly pyramidalized. Seven of the peptides were tested in vitro for their affinity for the µ-opioid receptor.

Moving out of CF3 -Land: Synthesis, Receptor Affinity, and in silico Studies of NK1 Receptor Ligands Containing a Pentafluorosulfanyl (SF5 ) Group.

The NK1 receptor (NK1R) is a molecular target for both approved and experimental drugs intended for a variety of conditions, including emesis, pain, and cancers. While contemplating modifications to the typical NK1R pharmacophore, we wondered whether the CF3 groups common for many NK1R ligands, could be replaced with some other moiety. Our attention was drawn by the SF5 group, and so we designed, synthesized, and tested ten novel SF5 -containing compounds for NK1R affinity. All analogues exhibit detectable NK1R binding, with the best of them, compound 5 a, (3-bromo-5-(pentafluoro-λ6 -sulfanyl)benzyl acetyl-L-tryptophanate) binding only slightly worse (IC50 =34.3 nM) than the approved NK1R-targeting drug, aprepitant (IC50 =27.7 nM). Molecular docking provided structural explanation of SAR. According to our analysis, the SF5 group in our compounds occupies a position similar to that of one of the CF3 groups of aprepitant as found in the crystal structure. Additionally, we checked whether the docking scoring function or energies derived from Fragment Molecular Orbital quantum chemical calculations may be helpful in explaining and predicting the experimental receptor affinities for our analogues. Both these methods produce moderately good results. Overall, this is the first demonstration of the utility of the SF5 group in the design of NK1R ligands.

Structure-activity relationship studies and biological properties evaluation of peptidic NRP-1 ligands: Investigation of N-terminal cysteine importance.

Neuropilin-1 (NRP-1) is a major co-receptor of vascular endothelial growth factor receptor-2 (VEGFR-2). It may also stimulate tumour growth and metastasis independently of VEGF-A165. These functions make VEGF-A165/NRP-1 complex formation and its inhibition of great interest, where NRP-1 is the target for which effective ligands are sought. Design of peptide-like inhibitors represent a strategy with great potential in the treatment of NRP-1-related disorders. Here, we present the synthesis, molecular modelling, structure-activity relationship studies as well as biological evaluation of peptides with the branched sequences H2N-X-Lys(hArg)-Dab-Oic-Arg-OH and H2N-Lys(X-hArg)-Dab-Oic-Arg-OH. Two of the designed peptides, in which Cys was inserted in X position, expressed high affinity (∼40 nM value) for NRP-1 and were resistant to enzymatic digestion in human serum. Moreover, peptide/NRP-1 complex promoted fast intracytoplasmic protein trafficking towards the plasma membrane in breast cancer cells. Our results suggest that these compounds might be good candidates for further development of VEGF-A165/NRP-1 inhibitors.

The influence of a synthetic growth hormone-releasing hormone analogue G11 and opioid peptide biphalin on selected fibroblasts parameters relevant to wound healing.

The treatment of hard-to-heal chronic wounds is still a major medical problem and an economic and social burden. In this work, we examine the proregenerative potential of two peptides, G11 (a trypsin-resistant analogue of growth hormone-releasing hormone [GHRH]) and biphalin (opioid peptide), and their combination in vitro on human fibroblasts (BJ). G11, biphalin and their combination exhibited no toxicity against BJ cells. On the contrary, these treatments significantly stimulated proliferation and migration of fibroblasts. Under inflammatory conditions (LPS-induced BJ cells), we noticed that the tested peptides decreased the levels of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and interleukin 1ß (IL-1ß). This was correlated with diminished phosphorylation levels of p38 kinase, but not those of ERK1/2. We found also that G11, biphalin and their combination activated the ERK1/2 signalling pathway, which has been previously implicated in promigratory activity of some regeneration enhancers, including opioids or GHRH analogues. Potential application of their combination requires further work, in particular in vivo experiments, in which the organism-level relevance of the discussed cell-level effects would be proven and, additionally, analgesic action of the opioid ingredient could be quantified.

Scandium-44 Radiolabeled Peptide and Peptidomimetic Conjugates Targeting Neuropilin-1 Co-Receptor as Potential Tools for Cancer Diagnosis and Anti-Angiogenic Therapy.

Pathological angiogenesis, resulting from an imbalance between anti- and pro-angiogenic factors, plays a pivotal role in tumor growth, development and metastasis. The inhibition of the angiogenesis process by the VEGF/VEGFR-2/NRP-1 pathway raises interest in the search for such interaction inhibitors for the purpose of the early diagnosis and treatment of angiogenesis-dependent diseases. In this work we designed and tested peptide-based radiocompounds that selectively bind to the neuropilin-1 co-receptor and prevent the formation of the pro-angiogenic VEGF-A165/NRP-1 complex. Three biomolecules, A7R and retro-inverso DR7A peptides, and the branched peptidomimetic Lys(hArg)-Dab-Pro-Arg (K4R), conjugated with macrocyclic chelator through two linkers' types, were labeled with theranostic scandium-44 radionuclide, and studied in vitro as potential targeted radiopharmaceuticals. ELISA (enzyme-linked immunosorbent assay) studies showed no negative effect of the introduced biomolecules' changes and high NRP-1 affinity in the case of A7R- and K4R-radiocompounds and a lack affinity for DR7A-radiocompounds. All radiopeptides showed a hydrophilic nature as well as high stability against ligand exchange reactions in cysteine/histidine solutions. Unfortunately, all radiocompounds showed unsatisfactory nano-scale stability in human serum, especially for use as therapeutic radioagents. Further work is ongoing and focused on the search for angiogenesis inhibitors that are more human serum stable.

Gαi-derived peptide binds the µ-opioid receptor.

BACKGROUND: G protein-coupled receptors (GPCRs) transduce external stimuli into the cell by G proteins via an allosteric mechanism. Agonist binding to the receptor stimulates GDP/GTP exchange within the heterotrimeric G protein complex, whereas recent structures of GPCR-G protein complexes revealed that the H5, S1 and S2 domains of Gα are involved in binding the active receptor, earlier studies showed that a short peptide analog derived from the C-terminus (H5) of the G protein transducin (Gt) is sufficient to stabilize rhodopsin in an active form. METHODS: We have used Molecular Dynamics simulations along with biological evaluation by means of radio-ligand binding assay to study the interactions between Gαi-derived peptide (G-peptide) and the µ-opioid receptor (µOR). RESULTS: Here, we show that a Gαi-derived peptide of 12 amino acids binds the µ-opioid receptor and acts as an allosteric modulator. The Gαi-derived peptide increases µOR affinity for its agonist morphine in a dose-dependent way. CONCLUSIONS: These results indicate that the GPCR-Gα peptide interaction observed so far for only rhodopsin can be extrapolated to µOR. In addition, we show that the C-terminal peptide of the Gαi subunit is sufficient to stabilize the active conformation of the receptor. Our approach opens the possibility to investigate the GPCR-G protein interface with peptide modification.

HPLC-PDA-ESI-HRMS-Based Profiling of Secondary Metabolites of Rindera graeca Anatomical and Hairy Roots Treated with Drought and Cold Stress.

To cope with environmental harmful conditions, plant cells developed adaptive strategy that involves production of a wide variety of complex secondary metabolites. The spectrum and quantity of biosynthesized compounds in specific plant species is determined by its genotype, tissue, developmental and physiological stage and environmental factors. This phenomenon was used to exploit the potential of anatomical and hairy root cultures of Rindera graeca to produce bioactive compounds. Cultivated in vitro roots were subjected to abiotic stresses i.e., drought or coldness. Next the extract profiling was performed using HPLC-PDA-ESI-HRMS method, as well quantitative determination of caffeic, rosmarinic and lithospermic B acids, that were present in all root extracts. Phenolic acids, flavonoids and iridoids represent the major groups of compounds detected in chemical profiles growing under various conditions roots. The highest number of phytochemicals was determined in roots subjected to coldness. Lithospermic B acid proved to be the most abundant compound in all investigated extracts. Among applied abiotic stress factors it was demonstrated that coldness affected to the most secondary metabolites production. The results of current study suggest that root cultures of R. graeca could serve as a new and abundant source of lithospermic B acid.

In Vitro Biological Evaluation of Aprepitant Based 177Lu-Radioconjugates.

Currently, the search for promising NK1R-positive tumor-targeting radiopharmaceuticals based on the structure of small molecular antagonists of neurokinin-1 receptor can be observed. Following this trend, we continued our evaluation of aprepitant-based 177Lu-radioconjugates in terms of future oncological applications. For this purpose, three novel aprepitant homologues were synthesized to broaden the previously obtained derivative portfolio, functionalized with the DOTA chelator and labeled with 68Ga and 177Lu. The newly evaluated radioconjugates showed the intended significant increase in lipophilicity compared to the previous ones, while maintaining stability in the human serum. Then, in a receptor binding study to the human NK1 receptor, we compared the two series of 177Lu-radioconjugates of aprepitant with each other and with the reference Substance P derivative currently used in glioblastoma therapy, clearly indicating the high affinity and better binding capacity of the novel radioconjugates. The in vitro experimental results included in the presented study, supported by labeling optimization, radioconjugate characterization and docking modeling of new aprepitant-derived radioagents, confirm our assumptions about the usefulness of aprepitant as a NK1R targeting vector and point out the perspectives for the forthcoming first in vivo trials.

Novel NK1R-Targeted 68Ga-/177Lu-Radioconjugates with Potential Application against Glioblastoma Multiforme: Preliminary Exploration of Structure-Activity Relationships.

Locoregionally administered, NK1 receptor (NK1R) targeted radionuclide therapy is a promising strategy for the treatment of glioblastoma multiforme. So far, the radiopharmaceuticals used in this approach have been based on the endogenous agonist of NK1R, Substance P or on its close analogues. Herein, we used a well-known, small molecular NK1R antagonist, L732,138, as the basis for the radiopharmaceutical vector. First, 14 analogues of this compound were evaluated to check whether extending the parent structure with linkers of different lengths would not deteriorate the NK1R binding. The tested analogues had affinity similar to or better than the parent compound, and none of the linkers had a negative impact on the binding. Next, five DOTA conjugates were synthesized and used for labelling with 68Ga and 177Lu. The obtained radioconjugates turned out to be fairly lipophilic but showed rather limited stability in human plasma. Evaluation of the receptor affinity of the (radio)conjugates showed that neither the chelator nor the metal negatively impacts the NK1R binding. The 177Lu-radioconjugates exhibited the binding characteristics towards NK1R similar or better than that of the 177Lu-labelled derivative of Substance P, which is in current clinical use. The experimental results presented herein, along with their structural rationalization provided by modelling, give insight for the further molecular design of small molecular NK1R-targeting vectors.

Synthesis, Physicochemical and Biological Study of Gallium-68- and Lutetium-177-Labeled VEGF-A165/NRP-1 Complex Inhibitors Based on Peptide A7R and Branched Peptidomimetic.

Neuropilin-1 (NRP-1) is a surface receptor found on many types of cancer cells. The overexpression of NRP-1 and its interaction with vascular endothelial growth factor-165 (VEGF165) are associated with tumor growth and metastasis. Therefore, compounds that block the VEGF165/NRP-1 interaction represent a promising strategy to image and treat NRP-1-related pathologies. The aim of the presented work was to design and synthesize radioconjugates of two known peptide-type inhibitors of the VEGF165/NRP-1 complex: A7R peptide and its shorter analog, the branched peptidomimetic Lys(hArg)-Dab-Pro-Arg. Both peptide-type inhibitors were coupled to a radionuclide chelator (DOTA) via a linker (Ahx) and so radiolabeled with Ga-68 and Lu-177 radionuclides, for diagnostic and therapeutic uses, respectively. The synthesized radioconjugates were tested for their possible use as theranostic-like radiopharmaceuticals for the imaging and therapy of cancers that overexpress NRP-1. The obtained results indicate good efficiency of the radiolabeling reaction and satisfactory stability, at least 3t1/2 for the 68Ga- and 1t1/2 for the 177Lu-radiocompounds, in solutions mimicking human body fluids. However, enzymatic degradation of both the studied inhibitors caused insufficient stability of the radiocompounds in human serum, indicating that further modifications are needed to sufficiently stabilize the peptidomimetics with inhibitory properties against VEGF165/NRP-1 complex formation.

Bifunctional Opioid/Melanocortin Peptidomimetics for Use in Neuropathic Pain: Variation in the Type and Length of the Linker Connecting the Two Pharmacophores.

Based on the mechanism of neuropathic pain induction, a new type of bifunctional hybrid peptidomimetics was obtained for potential use in this type of pain. Hybrids consist of two types of pharmacophores that are connected by different types of linkers. The first pharmacophore is an opioid agonist, and the second pharmacophore is an antagonist of the pronociceptive system, i.e., an antagonist of the melanocortin-4 receptor. The results of tests in acute and neuropathic pain models of the obtained compounds have shown that the type of linker used to connect pharmacophores had an effect on antinociceptive activity. Peptidomimetics containing longer flexible linkers were very effective at low doses in the neuropathic pain model. To elucidate the effect of linker lengths, two hybrids showing very high activity and two hybrids with lower activity were further tested for affinity for opioid (mu, delta) and melanocortin-4 receptors. Their complexes with the target receptors were also studied by molecular modelling. Our results do not show a simple relationship between linker length and affinity for particular receptor types but suggest that activity in neuropathic pain is related to a proper balance of receptor affinity rather than maximum binding to any or all of the target receptors.

The solid state VCD of a novel N-acylhydrazone trifluoroacetate.

A novel N-acylhydrazone with pharmaceutical importance was subject of structural and IR/VCD investigations in the solid state. In the crystal structure, dimers of anion-cation pairs are stabilized by H-bonding and ionic interactions. Some less common interaction types, like C=N···C-NH3+ (σ-hole) interactions, hydrazone-aromatic interactions and dispersive contacts of the CF3 groups are also present in the crystal. Satisfactory reproduction of the solid state IR and VCD spectra required that quantum-chemical calculations be done on a tetramer (four cation-anion pairs) cut out from the crystal structure, exhibiting key intermolecular interactions. Ten DFT functionals were assessed as to the agreement between the calculated and experimental spectra. Various approaches to scaling of the calculated frequencies were applied. The best results were yielded with individual (optimized) frequency scaling factors (FSFs) and band half-widths at half maximum-(HWHM) for four separate spectral subregions. The best matching between the experimental and theoretical spectra (according to SimIR, SimVCD and SimVDF indices) was found for the B3PW91 functional, however, a few other functionals follow closely in the ranking. Based on the quantum chemical calculations, spectral assignments have been made.

Biphalin-A Potent Opioid Agonist-As a Panacea for Opioid System-Dependent Pathophysiological Diseases?

Biphalin, one of the opioid agonists, is a dimeric analog of enkephalin with a high affinity for opioid receptors. Opioid receptors are widespread in the central nervous system and in peripheral neuronal and non-neuronal tissues. Hence, these receptors and their agonists, which play an important role in pain blocking, may also be involved in the regulation of other physiological functions. Biphalin was designed and synthesized in 1982 by Lipkowski as an analgesic peptide. Extensive further research in various laboratories on the antinociceptive effects of biphalin has shown its excellent properties. It has been demonstrated that biphalin exhibits an analgesic effect in acute, neuropathic, and chronic animal pain models, and is 1000 times more potent than morphine when administered intrathecally. In the course of the broad conducted research devoted primarily to the antinociceptive effect of this compound, it has been found that biphalin may also potentially participate in the regulation of other opioid system-dependent functions. Nearly 40 years of research on the properties of biphalin have shown that it may play a beneficial role as an antiviral, antiproliferative, anti-inflammatory, and neuroprotective agent, and may also affect many physiological functions. This integral review analyzes the literature on the multidirectional biological effects of biphalin and its potential in the treatment of many opioid system-dependent pathophysiological diseases.

A Phage Display-Identified Short Peptide Capable of Hydrolyzing Calcium Pyrophosphate Crystals-The Etiological Factor of Chondrocalcinosis.

Chondrocalcinosis is a metabolic disease caused by the presence of calcium pyrophosphate dihydrate crystals in the synovial fluid. The goal of our endeavor was to find out whether short peptides could be used as a dissolving factor for such crystals. In order to identify peptides able to dissolve crystals of calcium pyrophosphate, we screened through a random library of peptides using a phage display. The first screening was designed to select phages able to bind the acidic part of alendronic acid (pyrophosphate analog). The second was a catalytic assay in the presence of crystals. The best-performing peptides were subsequently chemically synthesized and rechecked for catalytic properties. One peptide, named R25, turned out to possess some hydrolytic activity toward crystals. Its catalysis is Mg2+-dependent and also works against soluble species of pyrophosphate.

The role of drugs and selected dietary factors in cutaneous squamous cell carcinogenesis.

Cutaneous squamous cell carcinoma represents the second most common non-melanoma skin cancer and its incidence increases worldwide. This review provides an overview of selected exogenous risk factors for cutaneous squamous cell carcinoma, which include drugs (azathioprine, calcineurin inhibitors, hydrochlorothiazide, angiotensin-converting-enzyme inhibitors) and few dietary factors (fat meet, whole milk products, arsenic) to better understand squamous skin cancer etiopathogenesis. Ingredients such as leafy vegetables, nuts, fish, caffeine, niacin are preventive factors for cutaneous squamous cell cancer. The heart transplant recipients have an increased risk of squamous cell carcinoma development than kidney or liver transplant ones and switching photosensitizing azathioprine to mycophenolate mofetil can reduce the incidence of cutaneous squamous cell carcinoma. The great attention should be paid to early change of cardiac photosensitizing antihypertensive drugs to non-photosensitizing ones among patients with a history of prior skin cancers and among organ transplant recipients. Based on current knowledge that ultra-violet radiation is the main risk factor for squamous cell carcinoma development, promotion of the skin self-examination, photoprotection, tanning bed avoidance and early skin cancer diagnosis is important for this tumour prevention.

Enantioseparation of ß2-amino acids by liquid chromatography using core-shell chiral stationary phases based on teicoplanin and teicoplanin aglycone.

Enantioseparation of nineteen ß2-amino acids has been performed by liquid chromatography on chiral stationary phases based on native teicoplanin and teicoplanin aglycone covalently bonded to 2.7 µm superficially porous silica particles. Separations were carried out in unbuffered (water/methanol), buffered [aqueous triethylammonium acetate (TEAA)/methanol] reversed-phase (RP) mode, and in polar-ionic (TEAA containing acetonitrile/methanol) mobile phases. Effects of pH in the RP mode, acid and salt additives, as well as counter-ion concentrations on chromatographic parameters have been studied. The structure of selectands (ß2-amino acids possessing aliphatic or aromatic side chains) and selectors (native teicoplanin or teicoplanin aglycone) was found to have a considerable influence on separation performance. Analysis of van Deemter plots and determination of thermodynamic parameters were performed to further explore details of the separation performance.

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.

The Role of VEGF Receptors as Molecular Target in Nuclear Medicine for Cancer Diagnosis and Combination Therapy.

One approach to anticancer treatment is targeted anti-angiogenic therapy (AAT) based on prevention of blood vessel formation around the developing cancer cells. It is known that vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptors (VEGFRs) play a pivotal role in angiogenesis process; hence, application of angiogenesis inhibitors can be an effective approach in anticancer combination therapeutic strategies. Currently, several types of molecules have been utilised in targeted VEGF/VEGFR anticancer therapy, including human VEGF ligands themselves and their derivatives, anti-VEGF or anti-VEGFR monoclonal antibodies, VEGF binding peptides and small molecular inhibitors of VEGFR tyrosine kinases. These molecules labelled with diagnostic or therapeutic radionuclides can become, respectively, diagnostic or therapeutic receptor radiopharmaceuticals. In targeted anti-angiogenic therapy, diagnostic radioagents play a unique role, allowing the determination of the emerging tumour, to monitor the course of treatment, to predict the treatment outcomes and, first of all, to refer patients for AAT. This review provides an overview of design, synthesis and study of radiolabelled VEGF/VEGFR targeting and imaging agents to date. Additionally, we will briefly discuss their physicochemical properties and possible application in combination targeted radionuclide tumour therapy.

Polyscias filicifolia (Araliaceae) Hairy Roots with Antigenotoxic and Anti-Photogenotoxic Activity.

Hairy root cultures are considered as a valuable source of bioactive phytoconstituents with expanding applicability for their production. In the present study, hairy root cultures of Polyscias filicifolia (Araliaceae), a traditional Southeast Asian medicinal plant, were established. The transformation with Agrobacterium rhizogenes ATCC 15834 allowed to obtain 15 root lines. The K-1 line, demonstrating the highest growth capabilities, was subjected to further investigations. To enhance the biosynthetic potential of hairy roots, methyl jasmonate elicitation approach was applied (MeJA; at different doses and exposure time), with subsequent transfer of elicited roots to control medium. This strategy resulted in chlorogenic acid production up to 1.59 mg/g dry weight. HPLC-PDA-ESI-MS analysis demonstrated variation in extracts composition and allowed to identify different caffeic and ferulic acid derivatives. Next, cytotoxic, antigenotoxic, and anti-photogenotoxic properties of hairy roots extracts were determined. None of the tested extracts were cytotoxic. In addition, they demonstrated significant antigenotoxic activity with the highest protective potential; up to 52% and 49% of inhibition of induction ratio (IR) induced by the 2-aminoanthracene was revealed for extracts derived from hairy roots elicited for 3 days with 50 µM MeJA and roots elicited for 7 days with 100 µM MeJA and then transferred for 30 days to control medium, respectively. These same extracts exhibited the highest anti-photogenotoxic potential, up to 36% of inhibition of chloropromazine-induced genotoxicity.

Novel bifunctional hybrid compounds designed to enhance the effects of opioids and antagonize the pronociceptive effects of nonopioid peptides as potent analgesics in a rat model of neuropathic pain.

ABSTRACT: The purpose of our work was to determine the role of nonopioid peptides derived from opioid prohormones in sensory hypersensitivity characteristics of neuropathic pain and to propose a pharmacological approach to restore the balance of these endogenous opioid systems. Nonopioid peptides may have a pronociceptive effect and therefore contribute to less effective opioid analgesia in neuropathic pain. In our study, we used unilateral chronic constriction injury (CCI) of the sciatic nerve as a neuropathic pain model in rats. We demonstrated the pronociceptive effects of proopiomelanocortin- and proenkephalin-derived nonopioid peptides assessed by von Frey and cold plate tests, 7 to 14 days after injury. The concentration of proenkephalin-derived pronociceptive peptides was increased more robustly than that of Met-enkephalin in the ipsilateral lumbar spinal cord of CCI-exposed rats, as shown by mass spectrometry, and the pronociceptive effect of one of these peptides was blocked by an antagonist of the melanocortin 4 (MC4) receptor. The above results confirm our hypothesis regarding the possibility of creating an analgesic drug for neuropathic pain based on enhancing opioid activity and blocking the pronociceptive effect of nonopioid peptides. We designed and synthesized bifunctional hybrids composed of opioid (OP) receptor agonist and MC4 receptor antagonist (OP-linker-MC4). Moreover, we demonstrated that they have potent and long-lasting antinociceptive effects after a single administration and a delayed development of tolerance compared with morphine after repeated intrathecal administration to rats subjected to CCI. We conclude that the bifunctional hybrids OP-linker-MC4 we propose are important prototypes of drugs for use in neuropathic pain.