A novel cinnamic and caffeic acid-conjugated peptide analogs with anticonvulsant and analgesic potency: Comparative analyses of trans/cis isomers.

The novel cinnamic acid (CA) (H4-CA, H5-CA, and H7-CA) and caffeic acid (KA) (H4-KA, H5-KA, and H7-KA) hemorphin analogs have recently been synthesized and their trans isomers have been tested for antiseizure and antinociceptive activity. In the present study, the cis forms of these compounds were tested and compared with their trans isomers in seizure and nociception tests in mice. The cis-H5-CA and H7-CA compounds showed efficacy against psychomotor seizures, whereas the trans isomers were ineffective. Both the cis and trans KA isomers were ineffective in the 6-Hz test. In the maximal electroshock (MES) test, the cis isomers showed superior antiseizure activity to the trans forms of CA and KA conjugates, respectively. The suppression of seizure propagation by cis-H5-CA and the cis-H5-KA was reversed by a kappa opioid receptor (KOR) antagonist. Naloxone and naltrindole were not effective. The cis-isomers of CA conjugates and cis-H7-KA produced significantly stronger antinociceptive effects than their trans-isomers. The cis-H5-CA antinociception was blocked by naloxone in the acute phase and by naloxone and KOR antagonists in the inflammatory phase of the formalin test. The antinociception of the KA conjugates was not abolished by opioid receptor blockade. None of the tested conjugates affected the thermal nociceptive threshold. The results of the docking analysis also suggest a model-specific mechanism related to the activity of the cis-isomers of CA and KA conjugates in relation to opioid receptors. Our findings pave the way for the further development of novel opioid-related antiseizure and antinociceptive therapeutics.

Potential anticonvulsant activity of novel VV-hemorphin-7 analogues containing unnatural amino acids: synthesis and characterization.

Herein, some new analogues of VV-hemorphin-7, modified at position 4 and 7 by the unnatural amino acids followed the structure Val-Val-Tyr-Xxx-Trp-Thr-Yyy-Arg-Phe-NH2, where Xxx is Ac5c (1-aminocyclopentanecarboxylic acid) or Ac6c (1-aminocyclohexane carboxylic acid) and Yyy is Dap (diaminopropanoic acid) or Dab (diaminobutanoic acid), were synthesized, characterized and investigated for anticonvulsant activity. The new synthetic peptide analogues were prepared by standard solid-phase peptide synthesis-Fmoc chemistry. A single intracerebroventricular (i.c.v.) injection at doses of 5, 10, and 20 µg/10 µl, respectively, was given before evaluation with timed intravenous pentylenetetrazole (ivPTZ) infusion test and 6-Hz psychomotor seizure test in mice. The acute neurological toxicity was determined using the rotarod test. To explain the structure-active properties of the modified peptides, some physicochemical characteristic was obtained. The FT-IR spectra and their second derivatives of the amide I, II, and III bands of the peptides show ß-sheet structure conformation. The calculation of isoelectric points, by potentiometric determination of dissociated constants, is in the range from 9.79 to 10.84. This study, for the first time, also reported on the reduction-oxidative potentials of the guanidine at Arg-moiety on such kind of peptides containing arginine and tyrosine residues in different medium and electrode surface. The VV-hemorphin-7 analogues 4 and 5 were the most active against the ivPTZ test, with the effect comparable to that of peptide 1 used as a positive control. Except compound 8, all other tested peptide analogues were ineffective to raise the threshold for the clonic seizures. The peptide analogue 5 showed 100% protection in the 6-Hz test, while the other seven VV-hemorphin-7 analogues have dose-dependent activity against psychomotor seizures comparable to 1. The novel peptides did not show neurotoxicity in the rotarod test.

Structure-activity relationship study on new hemorphin-4 analogues containing steric restricted amino acids moiety for evaluation of their anticonvulsant activity.

In the present study, several new analogues of hemorphin-4, modified with unnatural conformationally restricted amino acids followed the structure Aaa-Tyr-Xxx-Trp-Thr-NH2, where Aaa is the low-molecular-weight lipophilic adamantyl building block, and Xxx is Ac5c (1-aminocyclopentanecarboxylic acid) or Ac6c (1-aminocyclohexane carboxylic acid) was synthesized, characterized and investigated for anticonvulsant activity in three seizure tests, the maximal electroshock test (MES), 6-Hz psychomotor seizure test and timed intravenous pentylenetetrazole infusion (ivPTZ) test. The acute neurological toxicity was determined using the rota-rod test. The new synthetic neuropeptide analogues were prepared by solid-phase peptide synthesis-Fmoc chemistry and were evaluated in three doses of 1, 3 and 5 µg, respectively, administered intracerebroventricularly in male ICR mice. The physicochemical properties of these peptide analogues were evaluated as pKa and pI values were calculated using potentiometry. The IR spectrum of the compounds was recorded and the characteristic lines of both adamantane moiety and the peptide backbone were registered in the wavelength range from 4000 to 400 cm-1. The hexapeptide Ang IV was used as a positive control. From the six synthesized peptide analogues, the P4-5 was the most active at doses of 1 and 3 µg in the three seizure tests. The order of potency of other peptides was as follows: P4 > P4-3 = P4-4 > P4-2 > Ang IV in MES, P4-4 ≥ P4-1 > P4-3 > P4-2 > P4 > Ang IV in 6-Hz test and P4-4 = P4-3 > P4-2 = P4 > Ang IV in ivPTZ test. None of the peptides displayed neurotoxicity in the rota-rod test. Docking study results suggest that direct H-bonding and ionic interactions between our synthetic ligands and residues, responsible for coordination of Zn2+ along with hydrophobic interactions between our ligands and IRAP active site are the most important for the ligand binding. The results propose that incorporation of adamantane and cycloalkane building blocks in the peptide chain of the hemorphin-4 scaffold is important for the potential high biological activity.

Insights into the Interaction of LVV-Hemorphin-7 with Angiotensin II Type 1 Receptor.

Hemorphins are known for their role in the control of blood pressure. Recently, we revealed the positive modulation of the angiotensin II (AngII) type 1 receptor (AT1R) by LVV-hemorphin-7 (LVV-H7) in human embryonic kidney (HEK293) cells. Here, we examined the molecular binding behavior of LVV-H7 on AT1R and its effect on AngII binding using a nanoluciferase-based bioluminescence resonance energy transfer (NanoBRET) assay in HEK293FT cells, as well as molecular docking and molecular dynamics (MD) studies. Saturation and real-time kinetics supported the positive effect of LVV-H7 on the binding of AngII. While the competitive antagonist olmesartan competed with AngII binding, LVV-H7 slightly, but significantly, decreased AngII's kD by 2.6 fold with no effect on its Bmax. Molecular docking and MD simulations indicated that the binding of LVV-H7 in the intracellular region of AT1R allosterically potentiates AngII binding. LVV-H7 targets residues on intracellular loops 2 and 3 of AT1R, which are known binding sites of allosteric modulators in other GPCRs. Our data demonstrate the allosteric effect of LVV-H7 on AngII binding, which is consistent with the positive modulation of AT1R activity and signaling previously reported. This further supports the pharmacological targeting of AT1R by hemorphins, with implications in vascular and renal physiology.

Synthesis, characterization and anticonvulsant activity of new series of N-modified analogues of VV-hemorphin-5 with aminophosphonate moiety.

A new series of N-modified analogues of the VV-hemorphin-5 with aminophosphonate moiety have been synthesized, characterized and investigated for anticonvulsant activity. The novel peptide analogues were prepared by solid-phase peptide synthesis-Fmoc-strategy and were evaluated in the timed intravenous pentylenetetrazole infusion test (ivPTZ) and 6-Hz psychomotor seizure test in mice. The acute neurological toxicity was determined using the rotarod test. The redox potentials at glass carbonic electrode (GC) and the acid/base properties as pKa values of these peptide analogues were compared with the electrochemical behaviour of tyrosine- and tryptophan-containing peptides using different voltamperometric modes. Among the five tested peptide analogues, V3p was the most active against the ivPTZ test with effect comparable to that of the VV-hemorphin-5 (V1) used as a positive control. Dose-dependent elevation of the seizure threshold for myoclonic twitch and generalized clonic seizures was observed after i.c.v. administration of V2p, V4p and V5p as well as for forelimbs tonus in V4p peptides. The peptide analogues V2p-V5p were able to suppress dose-dependent psychomotor seizures in the 6-Hz test. In contrast, the V6p peptide showed either a pro-convulsant effect in the iv PTZ test or was inactive in the 6-Hz test. No changes in motor coordination were noted with the novel peptides. Docking study results suggest that kappa opioid receptor binding could be the mechanism of action of peptide derivatives with anticonvulsant activity. The results suggest that incorporation of aminophosphonate moiety at position 1 of the VV-hemorphin-5 scaffold deserve further evaluation in models of epilepsy and derivatization.

Anticonvulsant evaluation and docking analysis of VV-Hemorphin-5 analogues.

VV-Hemorphin-5 is an endogenous opioid peptide of the Hemorphin family with affinity at opioid receptors. A series of C-amide analogues have been synthesized, based on the structure of VV-Hemorphin-5, modified at position 1 and 7 by the un/natural amino acids (Aa8-Val-Val-Tyr-Pro-Trp-Thr-Gln-NH2 and Val-Val-Tyr-Pro-Trp-Thr-Aa1-NH2 ) using SPPS, Fmoc-chemistry. The peptide derivatives were evaluated for their anticonvulsant activity in three acute seizure tests in male ICR mice, the maximal electroshock (MES), the 6 Hz psychomotor seizure test, and the timed intravenous pentylenetetrazole (ivPTZ) infusion test. Their neurotoxicity was assessed in the rotarod test. Among the tested peptide analogues, V4 showed anticonvulsant activity in the three seizure tests that was comparable to the VV-Hemorphin-5 (V1) used as a positive control. While V5, V6, and V7 peptide derivatives exhibited anticonvulsant activity in the MES and 6 Hz test, they were inactive (V7) or showed pro-convulsant effect (V5 and V6) in the i.v. PTZ test. At a dose of 10 µg/mouse the peptide V2 was effective against clonic seizures induced by PTZ. Motor coordination was not affected by newly developed analogues of VV-Hemorphin-5. Docking study results suggest that kappa opioid receptor binding could be the mechanism of action of peptide derivatives with anticonvulsant activity. The results suggest that incorporation of nonproteinogenic and/or natural amino acids at position 1 and 7 of the VV-Hemorphin-5 scaffold deserve further evaluation in models of epilepsy and derivatization.

Synthesis, characterization and nociceptive screening of new VV-hemorphin-5 analogues.

In the present study, some new analogues of VV-hemorphin-5, modified at position 1 and 7 by the non-proteinogenic and/or natural amino acids followed the structures Xxx-Val-Val-Tyr-Pro-Trp-Thr-Gln-NH2 and Val-Val-Tyr-Pro-Trp-Thr-Yyy-NH2, where Xxx is Ile or Aib and Yyy is Lys/Orn/Dap/Dab were synthesized to investigate their potential antinociceptive activities. We report also the redox potentials and the acid/base properties as pKa values of these peptide analogues which were compared toward electrochemical behaviour of tryptophan containing peptides. All analogues showed a short lasting initial antinociceptive effect, however H2 hemorphin analogue is characterized with prolong and strong antinociceptive effect, while the other peptide analogues exerted more variable effects on the visceral nociception depending on the dose or time after the intracerebral injection.

Hemorphin-Based Analgesia: A Mechanism of Cupping Technique?

Background and Objective: Cupping is a time-honoured traditional healing modality for pain management and remains favoured by professionals and lay people across several cultures today. However, the analgesic mechanism of cupping is still poorly understood. In addition, clinical guidelines for standardized applications of cupping are currently lacking. The awareness of cupping marks has provoked curiosity about the connection between skin color changes and their benefit for local pain relief. Computer simulation is a promising approach for numerical modeling the cupping-evoked erythrocyte emigration. Quantitative proteomic profiling of cupping-induced blister fluid exhibited a significant decrease in the abundance of haemoglobin ß subunit. This finding provides a critical clue to paint a novel picture of the mechanism behind cupping. The hemorphins are a set of non-classical opioid peptides derived from the proteolysis of haemoglobin ß subunit. In the present study, a probable mechanism of hemorphin-based cupping analgesia is proposed. The hemorphin could also act as a potential biomarker for objective and timely quantitative clinical assessment of cupping in the management of pain conditions. A seminal theory may open a new avenue for future translational research on promoting the efficacy and safety of cupping analgesia. Conclusion: The local analgesic effect of cupping is probable in the context of haemoglobin degradation that bestows the appearance of hemorphins along with engaging opioid receptor signalling. Exploring the potential novel mechanism of cupping analgesia facilitates seeking non-pharmacologic pain interventions.

Metal-Peptide Complexes with Antimicrobial Potential for Cotton Fiber Protection.

A study of the formation of copper (II) complexes with hemorphin peptide motifs in alkalic water solutions is presented. The effect of the peptide ligand on the complexing properties of the Cu (II) ion was quantified by giving the stoichiometry and stability of the complex compounds in the medium in which they are formed using voltammetric (cyclic) and spectral (UV-Vis and fluorimetric) analytical techniques. The resulting complexes were examined via IR spectroscopy to detect M-N and M-O oscillations and using the EPR approach in solution and in the solid phase to view the coordination and ligand binding regime. The possibility of the synergistic action of copper ions in the antivirus protection processes of cotton fibers coated in the same solvent with the newly obtained complex compounds was also investigated. One of the advantages is the formation of the complexes in an environment where the immobilization takes place, which contributes to increasing the efficiency of the process. The obtained results may serve as an aid for future more detailed biological studies of structure-activity relationships (SARs).

Interaction of new VV-hemorphin-5 analogues with cell membrane models.

New analogues of the endogenous heptapeptide VV-hemorphin-5 (valorphin) synthesised by amino acid replacement allow for tailoring the peptide activity in vivo. Investigation of hemorphin-induced alterations of lipid bilayers' physicochemical parameters unravels membrane-mediated mechanisms of interaction with cells and subcellular structures. We studied the effect of modified valorphins with nociceptive activity on the structure, mechanical and electrical properties of lipid membrane models. Lower bending rigidity and higher specific capacitance of phosphatidylcholine bilayers were found in the presence of VV-hemorphin-5 analogues. Peptide partition constants for the transfer from the aqueous solution into the membrane were determined by isothermal titration calorimetry. It was found that the inclusion of non-proteinogenic acids with different number of methylene groups lead to alterations of hemorphin-membrane binding. The highest membrane affinity was obtained for a hemorphin derivative with dose-dependent variable effects on visceral nociception in mice. The valorphin analogue with the most pronounced anti-nociceptive effect in vivo induced the highest dipole and zeta potential change without significantly affecting the lipid packing at glycerol level in phosphatidylcholine bilayers.

Neuropeptides, substrates and inhibitors of human dipeptidyl peptidase III, experimental and computational study - A new substrate identified.

Dipeptidyl peptidase III (DPP III) is a cytosolic, two-domain zinc-exopeptidase. It is widely distributed in mammalian tissues, where it's involved in the final steps of normal intracellular protein degradation. However, its pronounced affinity for some bioactive peptides (angiotensins, enkephalins, and endomorphins) suggests more specific functions such as blood pressure regulation and involvement in pain regulation. We have investigated several different neuropeptides as potential substrates and inhibitors of human DPP III. The binding affinities and kinetic data determined by isothermal titration calorimetry, in combination with measurements of enzyme inhibition identified the hemorphin-related valorphin, tynorphin, S-tynorphin, and I-tynorphin as the most potent inhibitors of DPP III (actually slow substrates), whereas hemorphin-4 proved to be the best substrate of all neuropeptides examined. In addition, we have shown that the neuropeptides valorphin, Leu-valorphin-Arg, and the opioid peptide ß-casomorphin, are DPP III substrates. The molecular modelling of selected peptides shows uniform binding to the lower domain ß-strand residues of DPP III via peptide backbone atoms, but also previously unrecognized stabilizing interactions with conserved residues of the metal-binding site and catalytic machinery in the upper domain. The computational data helped explain the differences between substrates that are hydrolyzed effectively and those hydrolysed slowly by DPP III.

Valorphins alter physicochemical characteristics of phosphatidylcholine membranes: Datasets on lipid packing, bending rigidity, specific electrical capacitance, dipole potential, vesicle size.

Endogenous hemorphins are being intensively investigated as therapeutic agents in neuropharmacology, and also as biomarkers in mood regulation, inflammation and oncology. The datasets collected herein report physicochemical parameters of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes in the presence of VV-hemorphin-5 (Val-Val-Tyr-Pro-Trp-Thr-Gln) and analogues, modified at position 1 and 7 by the natural amino acid isoleucine or the non-proteinogenic 2-aminoisobutyric, 2,3-diaminopropanoic or 2,4-diaminobutanoic amino acids. These peptides have been previously screened for nociceptive activity and were chosen accordingly. The present article contains fluorescence spectroscopy data of Laurdan- and di-8-ANEPPS- labelled large unilamellar vesicles (LUV) providing the degree of hydration and dipole potential of lipid bilayers in the presence of VV-hemorphin-5 analogues. Lipid packing is accessible from Laurdan intensity profiles and generalized polarization datasets reported herein. The data presented on fluorescence intensity ratios of di-8-ANEPPS dye provide dipole potential values of phosphatidylcholine-valorphin membranes. Vesicle size and electrophoretic mobility datasets included refer to the effect of valorphins on the size distribution and ζ -potential of POPC LUVs. Investigation of physicochemical properties of peptides such as diffusion coefficients and heterogeneous rate constant relates to elucidation of transport mechanisms in living cells. Voltammetric data of valorphins are presented together with square-wave voltammograms of investigated peptides for calculation of their heterogeneous electron transfer rate constants. Datasets from the thermal shape fluctuation analysis of quasispherical 'giant' unilamellar vesicles (GUV) are provided to quantify the influence of hemorphin incorporation on the membrane bending elasticity. Isothermal titration calorimetric data on the thermodynamics of peptide-lipid interactions and the binding affinity of valorphin analogues to phosphatidylcholine membranes are reported. Data of frequency-dependent deformation of GUVs in alternating electric field are included together with the values of the specific electrical capacitance of POPC-valorphin membranes. The datasets reported in this article can underlie the formulation and implementation of peptide-based strategies in pharmacology and biomedicine.

LVV-hemorphin-7 (LVV-H7) plays a role in antinociception in a rat model of alcohol-induced pain disorders.

Alcohol can increase the sensitivity to painful stimulation or convert insensibility to pain at different stages. We hypothesized that chronic alcohol consumption changes the level of LVV-hemorphin-7 (abbreviated as LVV-H7, an opioid-like peptide generated from hemoglobin ß-chain), thereby affecting pain sensation. We established a chronic alcohol-exposed rat model to investigate the effects of LVV-H7. Adult male Sprague-Dawley rats were subjected to daily intraperitoneal injection of 10 % ethanol (w/v) at 0.5 g/kg for 15 days and subsequent alcohol withdrawal for 5 days. Using different pharmacological strategies to affect the LVV-H7 level, we investigated the correlation between LVV-H7 and pain-related behavior. Tail-flick and hot plate tests were employed to investigate alcohol-induced pain-related behavioral changes. The serum level of LVV-H7 was determined by ELISA. Our results showed that alcohol first induced an analgesia followed by a hyperalgesia during alcohol withdrawal, which could be driven by the quantitative change of LVV-H7. A positive correlation between the level of LVV-H7 and Δtail-flick latency (measured latency minus basal latency) confirmed this finding. Moreover, we revealed that the LVV-H7 levels were determined by the activity of cathepsin D and red blood cell/hemoglobin counts, which could be affected by alcohol. These results suggest that the deterioration of anti-nociception induced by alcohol is correlated to the decreased level of LVV-H7, and this could be due to alcohol-induced anemia. This study may help to develop LVV-H7 structure-based novel analgesics for treating alcohol-induced pain disorders and thus ameliorate the complications in alcoholics.

Antinociceptive Effects of VV-Hemorphin-5 Peptide Analogues Containing Amino phosphonate Moiety in Mouse Formalin Model of Pain.

BACKGROUND: Hemorphins are endogenous hemoglobin-derived peptides that belong to the family of "atypical" opioid peptides with both affinities to opioid receptors and ability to release other endogenous opioid peptides. OBJECTIVE: In the present study, peptide analogues of Valorphin (VV-hemorphin-5) containing amino phosphonate moiety synthesized by solid-phase peptide synthesis (Fmoc-strategy) were investigated for their potential antinociceptive activities and compared to the reference VV-H in formalin- induced model of acute and inflammatory pain in mice. METHODS: The hemorphin analogues were prepared by replacement of the one and/or two N-terminal Val in VV-hemorphin5 (VV-H) with ((dimethoxy phosphoryl) methyl)-L-valine and ((dimethoxy phosphoryl) methyl)-L-leucine to obtain the compounds pVV-H, pL-H, and pLV-H. Aiming to additionally prove the importance of amino acid valine, we introduced the ((dimethoxy phosphoryl) methyl)-L-leucine to the N-side of VV-hemorphin-5 (pLVV-H). The experiments were carried out on adult male ICR mice. All peptides were administered intracerebroventricularly at three doses (50, 25 and 12,5 µg/mouse). We have studied the effects of the peptides on acute (1st phase) and inflammatory (2nd phase) pain reaction using un experimental model with intraplantar formalin injection. RESULTS: VV-H showed a significant antinociceptive effect both in the acute and inflammatory phases of the test. Although Valorphin hexa-, hepta-, and octapeptide analogs demonstrated a significant antinociceptive effect, they showed substantial differences considering their effective dose and the phase of the test as compared to the Valorphin. DISCUSSION: Data showed that modified heptapeptides pVV-H and pLV-H exerted the same or better antinociception in acute and inflammatory pain, in comparison to the reference peptide, while pL-H and pLVV-H analogues were less effective. CONCLUSION: Our study contributes to the elucidation of the role of Valine and the number of amino acid residues in the structure of hemorphin peptide analogs in their effectiveness in suppressing both acute and inflammatory experimental pain.

Enhanced recombinant expression and purification of human IRAP for biochemical and crystallography studies.

Insulin-regulated aminopeptidase (IRAP) in humans is a membrane bound enzyme that has multiple functions. It was first described as a companion protein of the insulin-responsive glucose transporter, Glut4, in specialized vesicles. The protein has subsequently been shown to be identical to the oxytocinase/aminopeptidase or the angiotensin IV (Ang IV) receptor (AT4 receptor). Some AT4 ligand peptides, such as Ang IV and LVV-hemorphin-7, have been shown to act as IRAP inhibitors that exert memory-enhancing properties. As such IRAP has been a target for developing cognitive enhancers. To facilitate detailed mechanistic studies of IRAP catalysis and inhibition, and to pave the way for biophysical and structural studies of IRAP in complex with peptide inhibitors, we report here an optimized expression and purification system using High Five insect cells. We also report biochemical characterizations of the purified recombinant IRAP with a standard aminopeptidase substrate and an optimized IRAP peptide inhibitor with a Ki of 98 nM.

Behavioral effects evoked by the beta globin-derived nonapeptide LVV-H6.

LVV-hemorphin-6 (LVV-h6) is bioactive peptide and is a product of the degradation of hemoglobin. Since LVV-h6 effects are possibly mediated by opioid or AT4/IRAP receptors, we hypothesized that LVV-h6 would modify behavior. We evaluated whether LVV-h6 affects: i) anxiety-like behavior and locomotion; ii) depression-like behavior; iii) cardiovascular and neuroendocrine reactivity to emotional stress. Male Wistar rats ( ± 300 g) received LVV-h6 (153 nmol/kg i.p.) or vehicle (NaCl 0.9% i.p.). We used: i) open field (OF) test for locomotion; ii) elevated plus maze (EPM) for anxiety-like behavior; iii) forced swimming test (FST) for depression-like behavior and iv) air jet for cardiovascular and neuroendocrine reactivity to stress. Diazepam (2 mg/kg i.p.) and imipramine (15 mg/kg i.p.) were used as positive control for EPM and FST, respectively. To evaluate the LVV-h6 mechanisms, we used: the antagonist of oxytocin (OT) receptors (atosiban - ATS 1 and 0.1 mg/kg i.p.); the inhibitor of tyrosine hydroxylase (Alpha-methyl-p-tyrosine - AMPT 200 mg/kg i.p.) to investigate the involvement of catecholaminergic paths; and the antagonist of opioid receptors (naltrexone - NTX 0.3 mg/kg s.c.). We found that LVV-h6: i) evoked anxiolytic-like effect; ii) evoked antidepressant-like effect in the FST; and iii) did not change the locomotion, neuroendocrine and cardiovascular responses to stress. The LVV-h6 anxiolytic-like effect was not reverted by ATS and AMPT. However, the antidepressant effects were reverted only by NTX. Hence, our findings demonstrate that LVV-h6 modulates anxiety-like behavior by routes that are not oxytocinergic, catecholaminergic or opioid. The antidepressant-like effects of LVV-h6 rely on opioid pathways.

Positive Modulation of Angiotensin II Type 1 Receptor-Mediated Signaling by LVV-Hemorphin-7.

Hemorphins are hemoglobin ß-chain-derived peptides initially known for their analgesic effects via binding to the opioid receptors belonging to the family of G protein-coupled receptor (GPCR), as well as their physiological action on blood pressure. However, their molecular mechanisms in the regulation of blood pressure are not fully understood. Studies have reported an antihypertensive action via the inhibition of the angiotensin-converting enzyme, a key enzyme in the renin-angiotensin system. In this study, we hypothesized that hemorphins may also target angiotensin II (AngII) type 1 receptor (AT1R) as a key GPCR in the renin-angiotensin system. To investigate this, we examined the effects of LVV-hemorphin-7 on AT1R transiently expressed in human embryonic kidney (HEK293) cells using bioluminescence resonance energy transfer (BRET) technology for the assessment of AT1R/Gαq coupling and ß-arrestin 2 recruitment. Interestingly, while LVV-hemorphin-7 alone had no significant effect on BRET signals between AT1R and Gαq or ß-arrestin 2, it nicely potentiated AngII-induced BRET signals and significantly increased AngII potency. The BRET data were also correlated with AT1R downstream signaling with LVV-hemorphin-7 potentiating the canonical AngII-mediated Gq-dependent inositol phosphate pathway as well as the activation of the extracellular signal-regulated kinases (ERK1/2). Both AngII and LVV-hemorphin-7-mediated responses were fully abolished by AT1R antagonist demonstrating the targeting of the active conformation of AT1R. Our data report for the first time the targeting and the positive modulation of AT1R signaling by hemorphins, which may explain their role in the physiology and pathophysiology of both vascular and renal systems. This finding further consolidates the pharmacological targeting of GPCRs by hemorphins as previously shown for the opioid receptors in analgesia opening a new era for investigating the role of hemorphins in physiology and pathophysiology via the targeting of GPCR pharmacology and signaling.

Isolation of new ligands for orphan receptor MRGPRX1-hemorphins LVV-H7 and VV-H7.

The human MAS-related G protein-coupled receptor X1 (MRGPRX1) is a member of the GPCR family. The receptor is primate specific and expressed in the sensory neurons of dorsal root ganglion and trigeminal ganglion, where it is considered to be involved in the pain perception. The MRGPRX1 has unusual binding mechanism, as it is activated by several different ligands as well as several different fragments of precursor proteins. Thus, we hypothesize that it is activated by several unknown compounds as well since the receptor is still classified as orphan. Here, we describe the isolation of two novel endogenous ligands for the MRGPRX1 from human platelet preparation. The isolated ligands are hemoglobin ß-chain fragments, known members of the hemorphin family.

The hemoglobin derived peptide LVV-hemorphin-7 evokes behavioral effects mediated by oxytocin receptors.

LVV-hemorphin-7 (LVV-h7) is bioactive peptide resulting from degradation of hemoglobin ß-globin chain. LVV-h7 is a specific agonist of angiotensin IV receptor. This receptor belongs to the class of insulin-regulated aminopeptidases (IRAP), which displays oxytocinase activity. Herein, our aims were to assess whether: i) LVV-h7 modifies centrally organized behavior and cardiovascular responses to stress and ii) mechanisms underlying LVV-h7 effects involve activation of oxytocin (OT) receptors, probably as result of reduction of IRAP proteolytic activity upon OT. Adult male Wistar rats (270-370g) received (i.p.) injections of LVV-h7 (153nmol/kg), or vehicle (0.1ml). Different protocols were used: i) open field (OP) test for locomotor/exploratory activities; ii) Elevated Plus Maze (EPM) for anxiety-like behavior; iii) forced swimming test (FST) test for depression-like behavior and iv) air jet for cardiovascular reactivity to acute stress exposure. Diazepam (2mg/kg) and imipramine (15mg/kg) were used as positive control for EPM and FST, respectively. The antagonist of OT receptors (OTr), atosiban (1 and 0,1mg/kg), was used to determine the involvement of oxytocinergic paths. We found that LVV-h7: i) increased the number of entries and the time spent in open arms of the maze, an indicative of anxiolysis; ii) provoked antidepressant effect in the FS test; and iii) increased the exploration and locomotion; iv) did not change the cardiovascular reactivity and neuroendocrine responses to acute stress. Also, increases in locomotion and the antidepressant effects evoked by LVV-h7 were reverted by OTr antagonist. We conclude that LVV-h7 modulates behavior, displays antidepressant and anxiolytic effects that are mediated in part by oxytocin receptors.

Chronic administration of nandrolone increases susceptibility to morphine dependence without correlation with LVV-hemorphin 7 in rats.

LVV-hemorphin 7 (LVVYPWTQRF; LVV-H7), an N-terminal fragment of the ß-chain of hemoglobin cleaved by cathepsin D/pepsin, is an atypical endogenous opioid peptide that is found in high concentration in blood. LVV-H7 acts as a µ-opioid agonist and an inhibitor of insulin-regulated aminopeptidase. Subchronic administration of anabolic androgenic steroids (AAS) has been clinically proven to induce the synthesis of erythrocytes and increase hemoglobin concentrations. Patients with a history of AAS abuse are more susceptible to opioid abuse. We hypothesized that this association could be at least partially attributed to the sensitization of the mesocorticolimbic dopaminergic pathway by LVV-H7. Using the conditioned place preference test and neurochemical analysis, we investigated the possible mechanism underlying the effect of chronic nandrolone administration on morphine-induced reward and its correlation with LVV-H7 in rats. Either LVV-H7 may not sensitize the rewarding neural circuits or its inhibition on locomotor activity could mask reward-related behaviors. Chronic nandrolone pretreatment indeed caused a significant reward by low dose morphine, which did not cause any reward in control rats. However, coadministration of anti-LVV-H7 antiserum with nandrolone did not block this effect. This may rule out the possibility of the involvement of LVV-H7 in the action of nandrolone to intensify morphine-induced reward. Moreover, the serum level of LVV-H7 was mildly increased in response to chronic nandrolone administration in our animal model. According to the current clinical observations, we may conclude that the chronic administration of nandrolone can increase susceptibility to morphine dependence, but that this effect is not related to elevated LVV-H7.