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Type 2 diabetes is a chronic metabolic disorder characterized by high blood glucose levels due to either insufficient insulin production or ineffective utilization of insulin by the body. The enzyme dipeptidyl peptidase IV (DPP IV) plays a crucial role in degrading incretins that stimulate insulin secretion. Therefore, the inhibition of DPP IV is an established approach for the treatment of diabetes. Hemorphins are a class of short endogenous bioactive peptides produced by the enzymatic degradation of hemoglobin chains. Numerous in vitro and in vivo physiological effects of hemorphins, including DPP IV inhibiting activity, have been documented in different systems and tissues. However, the underlying molecular binding behavior of these peptides with DPP IV remains unknown. Here, computational approaches such as protein–peptide molecular docking and extensive molecular dynamics (MD) simulations were employed to identify the binding pose and stability of peptides in the active site of DPP IV. Findings indicate that hemorphins lacking the hydrophobic residues LVV and VV at the N terminal region strongly bind to the conserved residues in the active site of DPP IV. Furthermore, interactions with these critical residues were sustained throughout the duration of multiple 500 ns MD simulations. Notably, hemorphin 7 showed higher binding affinity and sustained interactions by binding to S1 and S2 pockets of DPP IV. [ABSTRACT FROM AUTHOR]

Valorphin (V1) is a naturally occurring peptide derived from hemoglobin that has been found to have an affinity for opioid receptors and exhibits antinociceptive and anticonvulsant activity. Some of its synthetic analogs containing an aminophosphonate moiety show structure-dependent potent antinociceptive effects. This study aimed to reveal a detailed picture of the antinociceptive mechanisms and behavioral effects of V1 and its recently synthesized phosphopeptide analog V2p in rodents using a range of methods. The studied peptides significantly reduced acute (mean V1–9.0, V2p–5.8 vs. controls–54.1 s) and inflammatory (mean V1–57.9 and V2p–53.3 vs. controls–107.6 s) nociceptive pain in the formalin test, as well as carrageenan-induced hyperalgesia (mean V1–184.7 and V2p–107.3 vs. controls–61.8 g) in the paw pressure test. These effects are mediated by activation of opioid receptors with a predominance of kappa in V1 antinociception and by delta, kappa, and mu receptors in V2p-induced antinociception. Both peptides did not change the levels of TNF-alpha and IL-1-beta in blood serum. V1 induces depression-like behavior, and V2p shows a tendency toward anxiolysis and short-term impairment of motor coordination without affecting exploratory behavior. The results characterize valorphin and its derivative as promising analgesics that exert their effects both centrally and peripherally, without causing severe behavioral changes in experimental animals. These encouraging data are a foundation for future studies focusing on the effects of hemorphins after long-term treatment. [ABSTRACT FROM AUTHOR]

Endogenous peptides have various modulating effects on many physiological and pathological processes, including the processing of pain information. Hemorphins are hemoglobin-derived peptides with affinity for both opioid receptors and insulin-regulated aminopeptidase. We recently reported on the antinociceptive effects of hemophins of different lengths and amino acid composition after their supraspinal injection. The present study aims to elucidate the potential effects of locally (intraplantar) injected hemorphin VV-H7 (Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe-NH2) and its analogs with one or two Arg-Gly-Asp sequence on carrageenan-induced hyperalgesia in rats. We used indomethacin as a reference anti-inflammatory analgesic. Pain thresholds and paw volume were measured before and at 1, 3, and 4 hours after intraplantar carrageenan injection using a paw pressure test and plethysmometry in male Wistar rats. The precursor peptide VV-H7 showed an antihyperalgesic effect comparable to that of indomethacin, while its peptide analogues one or two Arg-Gly-Asp sequence showed an acceleration of the antinociceptive effect, but shorter duration of antihyperalgesia and lack of efficacy against edema. [ABSTRACT FROM AUTHOR]

This manuscript presented the synthesis and characterization of two new N- and C-modified analogues of VV-hemorphin-7 containing RGD (Arg–Gly–Asp) residues as potential nociceptive agents and bioactive materials. It has been shown that the addition of one or two RGD sequences to natural VV-hemorphin-7 increases its effect on acute nociception, but the reduction of the inflammatory phase depends on the concentration of the peptide. The structure–property relationship of the new peptide derivatives was highlighted by electrochemical and FT-IR methods of analysis. Because of the proven bone-structural bonds of hydroxyapatite, the simultaneous deposition of peptide/hydroxyapatite on the surface of a titanium surface was investigated. The deposition was performed in a medium of gelatin solution containing dissolved amounts of peptide and hydroxyapatite using ultrasound. SEM–EDS analyzes confirmed the presence of a layer of the studied system. [ABSTRACT FROM AUTHOR]

Introduction: The evolving COVID-19 pandemic became a hallmark in human history, not only by changing lifestyles, but also by enriching scientific knowledge on viral infection and its consequences. Objective: Although the management of cardiorespiratory changes is pivotal to a favorable prognosis during severe clinical findings, dysregulation of other systems caused by SARS-CoV-2 infection may imbalance erythrocyte dynamics, such as a bidirectional positive feedback loop pathophysiology. Method and Results: Recent evidence shows that SARS-CoV-2 is capable of affecting the genetics and dynamics of erythrocytes and this coexists with a non-homeostatic function of cardiovascular, respiratory and renal systems during COVID-19. In hypothesis, SARS-CoV-2-induced systematical alterations of erythrocytes dynamics would constitute a setpoint for COVID-19-related multiple organ failure syndrome and death. Conclusion: The present review covers the most frequent erythrocyte-related non-homeostatic findings during COVID-19 capable of providing mechanistic clues of SARS-CoV-2-induced infection and inspiring therapeutic-oriented scientific evidence.

Valorphin (V1) is a naturally occurring peptide derived from hemoglobin that has been found to have an affinity for opioid receptors and exhibits antinociceptive and anticonvulsant activity. Some of its synthetic analogs containing an aminophosphonate moiety show structure-dependent potent antinociceptive effects. This study aimed to reveal a detailed picture of the antinociceptive mechanisms and behavioral effects of V1 and its recently synthesized phosphopeptide analog V2p in rodents using a range of methods. The studied peptides significantly reduced acute (mean V1–9.0, V2p–5.8 vs. controls–54.1 s) and inflammatory (mean V1–57.9 and V2p–53.3 vs. controls–107.6 s) nociceptive pain in the formalin test, as well as carrageenan-induced hyperalgesia (mean V1–184.7 and V2p–107.3 vs. controls–61.8 g) in the paw pressure test. These effects are mediated by activation of opioid receptors with a predominance of kappa in V1 antinociception and by delta, kappa, and mu receptors in V2p-induced antinociception. Both peptides did not change the levels of TNF-alpha and IL-1-beta in blood serum. V1 induces depression-like behavior, and V2p shows a tendency toward anxiolysis and short-term impairment of motor coordination without affecting exploratory behavior. The results characterize valorphin and its derivative as promising analgesics that exert their effects both centrally and peripherally, without causing severe behavioral changes in experimental animals. These encouraging data are a foundation for future studies focusing on the effects of hemorphins after long-term treatment.

Some new N- and C-modified biomolecular peptide analogues of both VV-hemorphin-5 and VV-hemorphin-7 with varied amino acids (Cys, Glu, His), 1-adamantanecarboxylic acid, and niacin (nicotinic acid) were synthesized by solid-phase peptide synthesis—Fmoc (9-fluorenylmethoxy-carbonyl) chemistry and were characterized in water solutions with different pH using spectroscopic and electrochemical techniques. Basic physicochemical properties related to the elucidation of the peptide structure at physiological pH have been also studied. The results showed that the interaction of peptide compounds with light and electricity preserves the structural and conformational integrity of the compounds in the solutions. Moreover, textile cotton fibers were modified with the new compounds and the binding of the peptides to the surface of the material was proved by FTIR and SEM analysis. Washing the material with an alkaline soap solution did not show a violation of the modified structure of the cotton. Antiviral activity against the human respiratory syncytial virus (HRSV-S2) and human adenovirus serotype 5 (HAdV-5), the antimicrobial activity against B. cereus and P. aeruginosa used as model bacterial strains and cytotoxic effect of the peptide derivatives and modified cotton textile material has been evaluated. Antimicrobial tests showed promising activity of the newly synthesized compounds against the used Gram-positive and Gram-negative bacteria. The compounds C-V, H-V, AC-V, and AH-V were found slightly more active than NH7C and NCH7. The activity has been retained after the deposition of the compounds on cotton fibers. [ABSTRACT FROM AUTHOR]

Follow-up of patients affected by COVID-19 has unveiled remarkable findings. Among the several sequelae caused by SARS-CoV-2 viral infection, it is particularly noteworthy that patients are prone to developing depression, anxiety, cognitive disorders, and dementia as part of the post-COVID-19 syndrome. The multisystem aspects of this disease suggest that multiple mechanisms may converge towards post-infection clinical manifestations. The literature provides mechanistic hypotheses related to changes in classical neurotransmission evoked by SARS-CoV-2 infection; nonetheless, the interaction of peripherally originated classical and non-canonic peptidergic systems may play a putative role in this neuropathology. A wealth of robust findings shows that hemoglobin-derived peptides are able to control cognition, memory, anxiety, and depression through different mechanisms. Early erythrocytic death is found during COVID-19, which would cause excess production of hemoglobin-derived peptides. Following from this premise, the present review sheds light on a possible involvement of hemoglobin-derived molecules in the COVID-19 pathophysiology by fostering neuroscientific evidence that supports the contribution of this non-canonic peptidergic pathway. This rationale may broaden knowledge beyond the currently available data, motivating further studies in the field and paving ways for novel laboratory tests and clinical approaches. [ABSTRACT FROM AUTHOR]

Follow-up of patients affected by COVID-19 has unveiled remarkable findings. Among the several sequelae caused by SARS-CoV-2 viral infection, it is particularly noteworthy that patients are prone to developing depression, anxiety, cognitive disorders, and dementia as part of the post-COVID-19 syndrome. The multisystem aspects of this disease suggest that multiple mechanisms may converge towards post-infection clinical manifestations. The literature provides mechanistic hypotheses related to changes in classical neurotransmission evoked by SARS-CoV-2 infection; nonetheless, the interaction of peripherally originated classical and non-canonic peptidergic systems may play a putative role in this neuropathology. A wealth of robust findings shows that hemoglobin-derived peptides are able to control cognition, memory, anxiety, and depression through different mechanisms. Early erythrocytic death is found during COVID-19, which would cause excess production of hemoglobin-derived peptides. Following from this premise, the present review sheds light on a possible involvement of hemoglobin-derived molecules in the COVID-19 pathophysiology by fostering neuroscientific evidence that supports the contribution of this non-canonic peptidergic pathway. This rationale may broaden knowledge beyond the currently available data, motivating further studies in the field and paving ways for novel laboratory tests and clinical approaches.

In the present study, a series of new analogues of both LVV- and VV-hemorphin-7 have been synthesized and characterized. They were modified at the N- and C-terminus with varied amino acids (Ile, D-Leu, D-Val, D-Phe) and enantiopure chiral S- and R- α-aminophosphonic acids ((dimethoxyphosphoryl)methyl)-valine and ((dimethoxyphosphoryl) methyl)-leucine) to optimize the physicochemical properties and to enhance their anticonvulsant potency. The novel peptide analogues were prepared by solid-phase peptide synthesis—Fmoc-strategy. Their structure–property relationship was studied by FT-IR spectroscopy and electrochemical methods. The lipophilicity is also presented. The anticonvulsant activity of peptide analogues, administered intracerebroventricularly, at doses of 1, 2.5, and 5 μg/10 μL, respectively, was explored by 6-Hz psychomotor seizure test, maximal electroshock test (MES) and a timed intravenous pentylenetetrazole (ivPTZ) infusion test in mice. The potential neurological toxicity of the substances was checked by a rotarod test. The H7 was used as a positive control. The H7-1 peptide analogue was the most active molecule against the psychomotor seizures, while H7-6 and H7-7 showed comparable to the positive group H7 potency in the MES test. The H7-5 to H7-8 analogues at the two tested doses of 2.5 and 5 μg/10 μl raised the threshold against ivPTZ-induced myoclonic, clonic, and tonic seizures. None of the hemorphin analogues exhibited neurotoxicity in the rotarod test. In conclusion, our results suggest that modified at N- and C-terminus of certain amino acids in the hemorphin analogues have a crucial role as a basis to design new LVV- and VV-hemorphin-7 analogues for experimental and clinical use. [ABSTRACT FROM AUTHOR]

Some new hemorphin-4 analogs with structures of Xxx-Pro-Trp-Thr-NH 2 and Tyr-Yyy-Trp-Thr-NH 2 , where Xxx is 2-amino-3-(4-hydroxy-2,6-dimethylphenyl)propanoic acid or 2-amino-3-(4-dibenzylamino-2,6-dimethylphenyl)propanoic acid, and Yyy is (2S,4S)-4-amino-pyrrolidine-2-carboxylic acid, were synthesized and characterized by electrochemical and spectral analyses. In vivo anticonvulsant and antinociceptive activities of peptide derivatives were studied after intracerebroventricular injection in mice. The therapeutic effects of the modified peptides on seizures and pain in mice were evaluated to provide valuable insights into the potential applications of the novel compounds. Electrochemical characterization showed that the compounds behave as weak protolytes and that they are in a soluble, stable molecular form at physiological pH values. The antioxidant activity of the peptides was evaluated with voltammetric analyses, which were confirmed by applying the 2,2-Diphenyl-1-picrylhydrazyl method. The compounds showed satisfactory results regarding their structural stability, reaching the desired centers for the manifestation of biological activity without hydrolysis processes at 37°C and physiological pH. Dm-H4 and H4-P1 exhibited 100% and 83% potency to suppress the psychomotor seizures in the 6-Hz test compared to 67% activity of H4. Notably, only the H4-P1 had efficacy in blocking the tonic component in the maximal electroshock test with a potency comparable to H4. All investigated peptides containing unnatural conformationally restricted amino acids showed antinociceptive effects. The analogs Db-H4 and H4-P1 showed the most pronounced and long-lasting effect in both experimental models of pain induced by thermal and chemical stimuli. Dm-H4 produced a dose-dependent thermal antinociception and H4-P2 inhibited only formalin-induced pain behavior., (© 2024 Deutsche Pharmazeutische Gesellschaft.)

The endogenous hemorphins are bioactive peptides with activity on opioid receptors. They are extensively studied and summarized in numerous reviews. During the last decade, several research teams have synthesized, characterized, and pharmacologically evaluated synthetic hemorphin analogs containing unusual amino acids, D-amino acids, α-aminophosphonic acids, and their derivatives. The present review summarizes the current studies on short-chain synthetic hemorphin peptide derivates containing non-natural amino acids. This review focuses on the structure–activity relationship analysis, details on specific methods for their characterization, and the advantage of synthetic hemorphin analogs compared to endogenous peptides as potent biologically active compounds with a complex mechanism of action.

Some new N- and C-modified biomolecular peptide analogues of both VV-hemorphin-5 and VV-hemorphin-7 with varied amino acids (Cys, Glu, His), 1-adamantanecarboxylic acid, and niacin (nicotinic acid) were synthesized by solid-phase peptide synthesis—Fmoc (9-fluorenylmethoxy-carbonyl) chemistry and were characterized in water solutions with different pH using spectroscopic and electrochemical techniques. Basic physicochemical properties related to the elucidation of the peptide structure at physiological pH have been also studied. The results showed that the interaction of peptide compounds with light and electricity preserves the structural and conformational integrity of the compounds in the solutions. Moreover, textile cotton fibers were modified with the new compounds and the binding of the peptides to the surface of the material was proved by FTIR and SEM analysis. Washing the material with an alkaline soap solution did not show a violation of the modified structure of the cotton. Antiviral activity against the human respiratory syncytial virus (HRSV-S2) and human adenovirus serotype 5 (HAdV-5), the antimicrobial activity against B. cereus and P. aeruginosa used as model bacterial strains and cytotoxic effect of the peptide derivatives and modified cotton textile material has been evaluated. Antimicrobial tests showed promising activity of the newly synthesized compounds against the used Gram-positive and Gram-negative bacteria. The compounds C-V, H-V, AC-V, and AH-V were found slightly more active than NH7C and NCH7. The activity has been retained after the deposition of the compounds on cotton fibers.

[Display omitted] • Some new caffeic and cinnamic acid-conjugated hemorphin derivatives were synthesized and characterized. • All peptides demonstrated anticonvulsant, antinociceptive, and antioxidant activity with low neurotoxicity. • Molecular docking, electrochemical, spectral, and fluorimetric analysis were studied. Based on the pharmacophore model of opioid receptors, our team recently synthesized a series of short‐chain hemorphin peptide analogs containing non‐natural amino acids. They demonstrated anticonvulsant and antinociceptive activity with low neurotoxicity. In the present study, a series of novel bioconjugates of N -modified hemorphin analogs containing second pharmacophore cinnamic acids (CA) or caffeic (KA) were synthesized by a traditional solid-phase Fmoc chemistry method for peptide synthesis. Electrochemical and fluorimetric analysis, in vivo anticonvulsant and antinociceptive activity in mice were conducted on the compounds. The three CA acid- (H4-CA, H5-CA, and H7-CA) and three KA acid- (H4-KA, H5-KA, and H7-KA) conjugated hemorphin derivatives exhibited potency at the highest doses of 2 µg/5 µl, administered by intracerebroventricular (icv) mode, against seizure spread in the maximal electroshock test (MES) in mice. The KA-conjugated H5-KA derivate, at the lowest dose, was the only compound that suppressed clonic seizures in the subcutaneous pentylenetetrazol (scPTZ) test. Except for the H5-CA, all tested CA acid- and KA acid-conjugated peptide derivates had the potency to increase the latency for clonic seizures in a dose-dependent mode. The activity against the psychomotor seizures in the 6-Hz test was detected only for the H4-CA (0.5 µg) and H4-KA (0.5 µg and 1 µg), respectively. All investigated peptides showed a more pronounced antinociceptive effect in the "intraplantar formalin" test compared to the "hot plate" test. Shorter chain analogs showed a better antinociceptive profile against tonic pain. The data suggest a DOR and KOR-mediated mechanism of action. According to the docking analysis, H7-CA showed a different antinociceptive profile than other investigated peptides. The novel peptide derivates did not exhibit neurotoxicity in the rotarod test. Our findings suggest that conjugated CA and KA morphine peptides can be used to develop novel morphine-related analogs with anticonvulsant and antinociceptive activity. [ABSTRACT FROM AUTHOR]

Here we report on the synthesis and characterization of three new N-modified analogues of hemorphin-4 with rhodamine B. Modified with chloroacetyl, chloride cotton fabric has been dyed and color coordinates of the obtained textile materials were determined. Antiviral and virucidal activities of both the peptide-rhodamine B compounds and the dyed textile material were studied. Basic physicochemical properties (acid-base behavior, solvent influence, kinetics) related to the elucidation of structural activity of the new modified peptides based on their steric open/closed ring effect were studied. The obtained results lead to the conclusion that in protic solvent with change in pH of the environment, direct control over the dyeing of textiles can be achieved. Both the new hybrid peptide compounds and the modification of functionalized textile materials with these bioactive hemorphins showed virucidal activity against the human respiratory syncytial virus (HRSV-S2) and human adenovirus serotype 5 (HAdV-5) for different time intervals (30 and 60 min) and the most active compound was Rh-3.

During the last three decades, a variety of different studies on bioactive peptides that are opioid receptor ligands, have been carried out, with regard to their isolation and identification, as well as their molecular functions in living organisms. Thus, in this review, we would like to summarize the present state-of-the art concerning hemorphins, methodological aspects of their identification, and their potential role as therapeutic agents. We have collected and discussed articles describing hemorphins, from their discovery up until now, thus presenting a very wide spectrum of their characteristic and applications. One of the major assets of the present paper is a combination of analytical and pharmacological aspects of peptides described by a team who participated in the initial research on hemorphins. This review is, in part, focused on the analysis of endogenous opioid peptides in biological samples using advanced techniques, description of the identification of synthetic/endogenous hemorphins, their involvement in pharmacology, learning, pain and other function. Finally, the part regarding hemorphin analogues and their synthesis, has been added.

Hemorphins are short peptides produced by the proteolysis of the beta subunit of hemoglobin. These peptides have diverse physiological effects especially in the nervous and the renin-angiotensin systems. Such effects occur through the modulation of a diverse range of proteins including enzymes and receptors. In this review, we focus on pharmacological and functional targeting of G protein-coupled receptors (GPCRs) by hemorphins and their implication in physiology and pathophysiology. Among GPCRs, the opioid receptors constitute the first set of targets of hemorphins with implication in analgesia. Subsequently, several other GPCRs have been reported to be directly or indirectly involved in hemorphins’ action. This includes the receptors for angiotensin II, oxytocin, bombesin, and bradykinin, as well as the human MAS-related G protein-coupled receptor X1. Interestingly, both orthosteric activation and allosteric modulation of GPCRs by hemorphins have been reported. This review links hemorphins with GPCR pharmacology and signaling, supporting the implication of GPCRs in hemorphins’ effects. Thus, this aids a better understanding of the molecular basis of the action of hemorphins and further demonstrates that hemorphin-GPCR axis constitutes a valid target for therapeutic intervention in different systems.

A novel analog of VV-hemorphin-5 containing azobenzene moiety has been synthesized and investigated for anticonvulsant activity in relation to its E → Z photophysical properties activated by long wavelength light at 365 nm. The synthesis was achieved by a modified SPPS by Fmoc-dimerization strategy. The electrochemical behavior before and after UV illumination was investigated using different voltammetric modes. The number of electrons transferred, heterogenic rate constant and diffusion coefficient for E- and Z-isomers were also evaluated. Revealing the governing principles involved in signaling and nerve pulse propagation requires the detailed characterization of the electrical properties of cell membranes. For probing the effect of synthesized azo-peptide on the membrane electrical properties, we measured the specific capacitance of lipid bilayers, representing a basic physical model of biomembranes with their simple reproducibility in laboratory conditions at controlled membrane composition and physicochemical parameters of the surrounding aqueous medium. Our results have shown reduced membrane capacitance in the presence of the azo-peptide, thus providing evidences for possible alterations in the dielectric permittivity of the bilayer. The (Val-Val-Tyr-Pro-Trp-Thr-Gln)2Azo peptide was explored also in vivo for preliminary anticonvulsant activity by using the 6-Hz seizure test and pentylenetetrazol (PTZ) seizure test in mice. The Z-isomer has exhibited higher potency compared to E-isomer most pronouncedly in the 6 Hz test for psychomotor seizures where the compound had activity at all three tested doses. It was found that the Z-isomer decrease the latency for onset of clonic seizures induced by PTZ. These results demonstrate that the Z-isomer deserves further evaluation in other screening tests for anticonvulsant activity. [ABSTRACT FROM AUTHOR]

Angiotensin-I converting enzyme (ACE) is a zinc metallopeptidase that has an important role in regulating the renin-angiotensin-aldosterone system (RAAS). It is also an important drug target for the management of cardiovascular diseases. Hemorphins are endogenous peptides that are produced by proteolytic cleavage of beta hemoglobin. A number of studies have reported various therapeutic activities of hemorphins. Previous reports have shown antihypertensive action of hemorphins via the inhibition of ACE. The sequence of hemorphins is highly conserved among mammals, except in camels, which harbors a unique Q>R variation in the peptide. Here, we studied the ACE inhibitory activity of camel hemorphins (LVVYPWTRRF and YPWTRRF) and non-camel hemorphins (LVVYPWTQRF and YPWTQRF). Computational methods were used to determine the most likely binding pose and binding affinity of both camel and non-camel hemorphins within the active site of ACE. Molecular dynamics simulations showed that the peptides interacted with critical residues in the active site of ACE. Notably, camel hemorphins showed higher binding affinity and sustained interactions with all three subsites of the ACE active site. An in vitro ACE inhibition assay showed that the IC50 of camel hemorphins were significantly lower than the IC50 of non-camel hemorphins.

A qualitative study is presented, where the main question was whether food-derived hemorphins, i.e., originating from digested alimentary hemoglobin, could pass the intestinal barrier and/or the blood–brain barrier (BBB). Once absorbed, hemorphins are opioid receptor (OR) ligands that may interact with peripheral and central OR and have effects on food intake and energy balance regulation. LLVV-YPWT (LLVV-H4), LVV-H4, VV-H4, VV-YPWTQRF (VV-H7), and VV-H7 hemorphins that were previously identified in the 120 min digest resulting from the simulated gastrointestinal digestion of hemoglobin have been synthesized to be tested in in vitro models of passage of IB and BBB. LC-MS/MS analyses yielded that all hemorphins, except the LLVV-H4 sequence, were able to cross intact the human intestinal epithelium model with Caco-2 cells within 5–60 min when applied at 5 mM. Moreover, all hemorphins crossed intact the human BBB model with brain-like endothelial cells (BLEC) within 30 min when applied at 100 µM. Fragments of these hemorphins were also detected, especially the YPWT common tetrapeptide that retains OR-binding capacity. A cAMP assay performed in Caco-2 cells indicates that tested hemorphins behave as OR agonists in these cells by reducing cAMP production. We further provide preliminary results regarding the effects of hemorphins on tight junction proteins, specifically here the claudin-4 that is involved in paracellular permeability. All hemorphins at 100 µM, except the LLVV-H4 peptide, significantly decreased claudin-4 mRNA levels in the Caco-2 intestinal model. This in vitro study is a first step toward demonstrating food-derived hemorphins bioavailability which is in line with the growing body of evidence supporting physiological functions for food-derived peptides.

Two series of new VV-hemorphin-5 analogs with structures Val-Val-Tyr-Xxx-Trp-Thr-Gln-NH 2 and Adam-Val-Val-Tyr-Xxx-Trp-Thr-Gln-NH 2 , where Xxx is Ac5c (1-aminocyclopentane-1-carboxylic acid), Ac6c (1-aminocyclohexane-1-carboxylic acid), Ac7c (1-aminocycloheptane-1-carboxylic acid), and Adam is the low-molecular-weight lipophilic adamantyl building block, were synthesized, characterized electrochemically and evaluated for antioxidant, anti-hyperalgesia, and anticonvulsant activity. The design of the compounds followed the strategy to improve the propensity for aqueous solubility and/or to increase their affinity for the target receptor or enzyme. The partition coefficient value shows that the peptide scaffold goes from hydrophilic to lipophilic with the increasing size of the cycloalkane ring and even more with the introduction of the adamantane. The peptides C5-V and C7-V were the only analogs that provoked an immediate antinociceptive effect changing the mechanical pain threshold. The six new peptide analogs produced a significant and long-lasting carrageenan model of inflammatory pain in rats. While the adamantane hemorphin analog Ad7-V was the only compound with the potency to suppress psychomotor seizures in the 6-Hz test, the C6-V and Ad6-V exhibited protective activity against the seizure spread in the maximal electroshock seizure test in mice. The active analogs did not show neurotoxicity or sedative effects. Our results revealed a structure-related specific activity of a newly designed hemorphin analog that could be used as a template for future modification and preparation of compounds with potential analgesic and anticonvulsant activity., (© 2023 Deutsche Pharmazeutische Gesellschaft.)

Proteomic surveys with top-down platforms are today revealing thousands of naturally occurring fragments of bigger proteins. Some of them have not functional meaning because they derive from pathways responsible for protein degradation, but many have specific functions, often completely different from that one of the parent proteins. These peptides encrypted in the protein sequence are nowadays called cryptides. They are frequent in the animal and plant kingdoms and represent a new interesting -omic field of investigation. To point out how much widespread is their presence, we describe here the most studied cryptides from very common sources such as serum albumin, immunoglobulins, hemoglobin, and from saliva and milk proteins. Given its vastness, it is unfeasible to cover the topic exhaustively, therefore only several selected examples of cryptides from other sources are thereafter reported. Demanding is the development of new -omic platforms for the functional screening of new cryptides, which could provide suggestion for peptides and peptido-mimetics with variegate fields of application. [ABSTRACT FROM AUTHOR]

A qualitative study is presented, where the main question was whether food-derived hemorphins, i.e., originating from digested alimentary hemoglobin, could pass the intestinal barrier and/or the blood--brain barrier (BBB). Once absorbed, hemorphins are opioid receptor (OR) ligands that may interact with peripheral and central OR and have effects on food intake and energy balance regulation. LLVV-YPWT (LLVV-H4), LVV-H4, VV-H4, VV-YPWTQRF (VV-H7), and VV-H7 hemorphins that were previously identified in the 120 min digest resulting from the simulated gastrointestinal digestion of hemoglobin have been synthesized to be tested in in vitro models of passage of IB and BBB. LC-MS/ MS analyses yielded that all hemorphins, except the LLVV-H4 sequence, were able to cross intact the human intestinal epithelium model with Caco-2 cells within 5-60 min when applied at 5 mM. Moreover, all hemorphins crossed intact the human BBB model with brain-like endothelial cells (BLEC) within 30 min when applied at 100 μM. Fragments of these hemorphins were also detected, especially the YPWT common tetrapeptide that retains OR-binding capacity. A cAMP assay performed in Caco-2 cells indicates that tested hemorphins behave as OR agonists in these cells by reducing cAMP production. We further provide preliminary results regarding the effects of hemorphins on tight junction proteins, specifically here the claudin-4 that is involved in paracellular permeability. All hemorphins at 100 μM, except the LLVV-H4 peptide, significantly decreased claudin-4 mRNA levels in the Caco-2 intestinal model. This in vitro study is a first step toward demonstrating food-derived hemorphins bioavailability which is in line with the growing body of evidence supporting physiological functions for food-derived peptides. [ABSTRACT FROM AUTHOR]

The endogenous hemorphins are bioactive peptides with activity on opioid receptors. They are extensively studied and summarized in numerous reviews. During the last decade, several research teams have synthesized, characterized, and pharmacologically evaluated synthetic hemorphin analogs containing unusual amino acids, D-amino acids, α-aminophosphonic acids, and their derivatives. The present review summarizes the current studies on short-chain synthetic hemorphin peptide derivates containing non-natural amino acids. This review focuses on the structure-activity relationship analysis, details on specific methods for their characterization, and the advantage of synthetic hemorphin analogs compared to endogenous peptides as potent biologically active compounds with a complex mechanism of action.

Abstract: A CE-tandem MS method was optimised and validated for selective and specific determination of LVV- and VV-hemorphin-7 peptides in cerebrospinal fluid. These two small peptides originate from haemoglobin beta chains. They possess relevant biological activity and recently a potential biomarker role in posterior cranial fossa paediatric brain tumour disease was evidenced. The separation was optimised using formic acid as background electrolyte and a water/methanol mixture, containing 0.1% (v/v) formic acid, as sheath liquid. The two peptides, differing in only one amino acid of the sequence at the N-terminal side were baseline separated in less than 15min. The method allowed a very reduced and rapid sample pretreatment and was successfully applied to hemorphins determination in patient samples without matrix interferences. The method successfully passed bioanalytical validation showing linearity, accuracy and precision data on cerebrospinal fluid matrix within the acceptable values. The analysis of cerebrospinal fluid of patients affected by different posterior cranial fossa tumour forms confirmed our previous findings showing the absence of hemorphins in the pre-surgical cerebrospinal fluid and their presence in the post-ones and controls. The present method saves costs and time due to capillary electrophoresis miniaturisation and to the absence of chromatographic column and gradient elution and allows numerous injections per sample starting from few microlitres of cerebrospinal fluid. [Copyright &y& Elsevier]

Thalassemia major continues to be a significant health problem for Mediterranean, Afro-Arabic countries, India and South Easth Asia. It was generally assumed that the β-thalassemia heterozygotes do not bear significant medical risks except a mild microcytic anemia. Nonetheless, increasing number of reports associate β-thalassemia trait with autoimmune conditions, nephritis, diabetes, arthritis, fibromyalgia and asthma. Available sparse data indicate reduced incidence of systemic lupus erythematosus (SLE) in β-thalassemia heterozygotes; yet, if two conditions coexist, the SLE manifestations occur much severer. These associations make sense when considering that the hemoglobin β-chain locus at 11p15.5 resides in close proximity to eight genes with profound roles in immune regulation: STIM1, CD151, TC21/RRAS2, SIGIRR/TOLL/IL1R8, pp52/LSP1 (lymphocyte specific protein), TRIM21, toll interacting protein (TOLLIP) and SLEN3. β-Thalassemia trait accompaniment to autoimmune disease may be the result of haplotypal associations between the close proximity genes. An alternative explanation to thalassemia heterozygosity: autoimmune disease association may be the changed concentrations of hemorphins. Hemorphins are endogenous opioid peptides derived via proteolytical cleavage of hemoglobin. They are shown to bind diverse opioid receptors and act anti-inflammatory. Their reduced expression in thalassemia heterozygosity may explain a proinflammatory stage and autoimmunity vulnerability. [ABSTRACT FROM AUTHOR]

Abstract: Previous studies showed a significantly reduced level of hemorphins in the serum of diabetes patients. In order to elucidate the biochemical mechanisms responsible for this anomaly, the influence of hemoglobin glycation on hemorphin generation was studied. The glycation of hemoglobin occurs in the blood of diabetes patients and this could modify its enzymatic digestion and the resulting proteolytic products. Several samples of hemoglobin were obtained from the blood of type 1 diabetes patients (n =8) and normal healthy control subjects (n =2). The glycated hemoglobin samples were classified on the basis of their HbA1c values expressed as a percentage of total hemoglobin. Four solutions of glycated hemoglobin characterized by HbA1c values of 6%, 9.1%, 10.7% and 12.1% were treated with cathepsin D and the hemorphins obtained following the proteolysis were compared to controls. It was found that hemorphins were produced whatever the level of glycation of hemoglobin and also that the degree of glycation had no effect on the quantity of hemorphins released. Thus the alteration of hemoglobin does not seem to be the essential reason for the decrease in hemorphin concentrations in the sera of diabetic patients. [Copyright &y& Elsevier]

Abstract: Hemorphins are multifunctional peptides derived from hemoglobin or blood processing. They have been found at high levels within the central nervous system where they have a direct effect on neuronal cells via peptidergic receptors. As relatively few studies have examined their metabolic stability in the brain, such investigation was performed to locate the cellular distribution of enzymatic activity against these peptides. High-performance liquid chromatography (HPLC) combined with electrospray ionisation mass spectrometry (ESI-MS) allows identification of degradation products resulting from incubation of hemorphin-7 peptides (LVV-hemorphin-7, VV-hemorphin-7 and hemorphin-7) with subcellular fractions isolated from rat brain tissue. Metabolic activities were found against the three peptides in brain homogenate and subcellular fractions with the highest metabolic activity (<3% peptide remaining after 10min) observed in the microsomal fraction which processed hemorphin-7 peptides mainly into N-terminal fragments (giving LVVH5) suggesting action of brain-membrane enzymes with C-terminal specificity. Incubation of the ACE inhibitor captopril (0.2μM) with microsomal fraction, together with LVVH7, decreased the processing of LVVH7 to form LVVH5 by 85%. [Copyright &y& Elsevier]

The human hemorphin LVV-H7 is an endogenous cleavage product of the hemoglobin β, γ, ε or δ chain exhibiting potential pharmaceutical relevance for blood pressure regulation, the treatment of Alzheimer’s disease or learning deficiencies. Here we present the development of a multi-component capillary zone electrophoretic method (CZE-UV), allowing the simultaneous quantification of LVV-H7 and four N-terminal degradation products generated in EDTA plasma. Hemorphins in the supernatant of precipitated plasma samples are quantified by external calibration. Validation of the procedure oriented towards international pharmaceutical guidelines and demonstrated excellent linearity ( r 2 ≥0.999), good precision (repeatability and reproducibility below 11%), accuracy (−8.4%–4%), ruggedness and an appropriate lower limit of quantification (LLOQ 1.0 μg mL−1). This procedure was applied to stability studies of LVV-H7 in human EDTA plasma attended by profiling metabolites using qualitative MALDI-TOF MS analysis. We detected the activity of a soluble plasma form of aminopeptidase M causing successive N-terminal truncation. This is the first time that LVV-H7 degradation as well as its metabolite production have systematically been monitored by a quantitative CZE-UV procedure, underlining the growing importance of such techniques in peptide analysis. In addition, our results give useful hints for future drug development of LVV-H7. [ABSTRACT FROM AUTHOR]

The role of angiotensin IV (AngIV) in the regulation of angiotensin-converting enzyme (ACE) was studied in vitro. This study demonstrates that this active fragment appeared as a novel endogenous ACE inhibitor. Inhibitory kinetic studies revealed that AngIV acts as a purely competitive inhibitor with a Ki value of 35 μM. AngIV was found to be quite resistant to ACE hydrolysis opposite to hemorphins which are both ACE inhibitors and substrates. In order to confirm a putative role of AngIV and hemorphins in the Renin–Angiotensin system (RAS) regulation, we studied their influence on AngI conversion. We noticed that 16.7 μM of both peptides decreased more than 50% of AngI conversion to AngII in vitro. The capacity of hemorphins, particularly LVVH-7, and AngIV to inhibit ACE activity here suggests a synergistic relation between these two peptides and the regulation of RAS. [Copyright &y& Elsevier]

Introduction: The evolving COVID-19 pandemic became a hallmark in human history, not only by changing lifestyles, but also by enriching scientific knowledge on viral infection and its consequences., Objective: Although the management of cardiorespiratory changes is pivotal to a favorable prognosis during severe clinical findings, dysregulation of other systems caused by SARS-CoV-2 infection may imbalance erythrocyte dynamics, such as a bidirectional positive feedback loop pathophysiology., Method and Results: Recent evidence shows that SARS-CoV-2 is capable of affecting the genetics and dynamics of erythrocytes and this coexists with a non-homeostatic function of cardiovascular, respiratory and renal systems during COVID-19. In hypothesis, SARS-CoV-2-induced systematical alterations of erythrocytes dynamics would constitute a setpoint for COVID-19-related multiple organ failure syndrome and death., Conclusion: The present review covers the most frequent erythrocyte-related non-homeostatic findings during COVID-19 capable of providing mechanistic clues of SARS-CoV-2-induced infection and inspiring therapeutic-oriented scientific evidence., Competing Interests: The authors declare no conflicts of interest., (© 2022 Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular. Published by Elsevier España, S.L.U.)

Here we report on the synthesis and characterization of three new N-modified analogues of hemorphin-4 with rhodamine B. Modified with chloroacetyl, chloride cotton fabric has been dyed and color coordinates of the obtained textile materials were determined. Antiviral and virucidal activities of both the peptide-rhodamine B compounds and the dyed textile material were studied. Basic physicochemical properties (acid-base behavior, solvent influence, kinetics) related to the elucidation of structural activity of the new modified peptides based on their steric open/closed ring effect were studied. The obtained results lead to the conclusion that in protic solvent with change in pH of the environment, direct control over the dyeing of textiles can be achieved. Both the new hybrid peptide compounds and the modification of functionalized textile materials with these bioactive hemorphins showed virucidal activity against the human respiratory syncytial virus (HRSV-S2) and human adenovirus serotype 5 (HAdV-5) for different time intervals (30 and 60 min) and the most active compound was Rh-3. [ABSTRACT FROM AUTHOR]

During the last three decades, a variety of different studies on bioactive peptides that are opioid receptor ligands, have been carried out, with regard to their isolation and identification, as well as their molecular functions in living organisms. Thus, in this review, we would like to summarize the present state-of-the art concerning hemorphins, methodological aspects of their identification, and their potential role as therapeutic agents. We have collected and discussed articles describing hemorphins, from their discovery up until now, thus presenting a very wide spectrum of their characteristic and applications. One of the major assets of the present paper is a combination of analytical and pharmacological aspects of peptides described by a team who participated in the initial research on hemorphins. This review is, in part, focused on the analysis of endogenous opioid peptides in biological samples using advanced techniques, description of the identification of synthetic/endogenous hemorphins, their involvement in pharmacology, learning, pain and other function. Finally, the part regarding hemorphin analogues and their synthesis, has been added. [ABSTRACT FROM AUTHOR]

Hemorphins are short peptides produced by the proteolysis of the beta subunit of hemoglobin. These peptides have diverse physiological effects especially in the nervous and the renin-angiotensin systems. Such effects occur through the modulation of a diverse range of proteins including enzymes and receptors. In this review, we focus on pharmacological and functional targeting of G protein-coupled receptors (GPCRs) by hemorphins and their implication in physiology and pathophysiology. Among GPCRs, the opioid receptors constitute the first set of targets of hemorphins with implication in analgesia. Subsequently, several other GPCRs have been reported to be directly or indirectly involved in hemorphins' action. This includes the receptors for angiotensin II, oxytocin, bombesin, and bradykinin, as well as the human MAS-related G protein-coupled receptor X1. Interestingly, both orthosteric activation and allosteric modulation of GPCRs by hemorphins have been reported. This review links hemorphins with GPCR pharmacology and signaling, supporting the implication of GPCRs in hemorphins' effects. Thus, this aids a better understanding of the molecular basis of the action of hemorphins and further demonstrates that hemorphin-GPCR axis constitutes a valid target for therapeutic intervention in different systems. [ABSTRACT FROM AUTHOR]

Hemorphins are endogenous peptides, 4–10 amino acids long, belonging to the family of atypical opioid peptides released during the sequential cleavage of hemoglobin protein. Hemorphins have been shown to exhibit diverse therapeutic effects in both human and animal models. However, the precise cellular and molecular mechanisms involved in such effects remain elusive. In this review, we summarize and propose potential mechanisms based on studies that investigated the biological activity of hemorphins of different lengths on multiple therapeutic targets. Special emphasis is given to molecular events related to renin-angiotensin system (RAS), opioid receptors and insulin-regulated aminopeptidase receptor (IRAP). This review provides a comprehensive coverage of the molecular mechanisms that underpin the therapeutic potential of hemorphins. Furthermore, it highlights the role of various hemorphin residues in pathological conditions, which could be explored further for therapeutic purposes. [ABSTRACT FROM AUTHOR]

Angiotensin-I converting enzyme (ACE) is a zinc metallopeptidase that has an important role in regulating the renin-angiotensin-aldosterone system (RAAS). It is also an important drug target for the management of cardiovascular diseases. Hemorphins are endogenous peptides that are produced by proteolytic cleavage of beta hemoglobin. A number of studies have reported various therapeutic activities of hemorphins. Previous reports have shown antihypertensive action of hemorphins via the inhibition of ACE. The sequence of hemorphins is highly conserved among mammals, except in camels, which harbors a unique Q>R variation in the peptide. Here, we studied the ACE inhibitory activity of camel hemorphins (LVVYPWTRRF and YPWTRRF) and non-camel hemorphins (LVVYPWTQRF and YPWTQRF). Computational methods were used to determine the most likely binding pose and binding affinity of both camel and non-camel hemorphins within the active site of ACE. Molecular dynamics simulations showed that the peptides interacted with critical residues in the active site of ACE. Notably, camel hemorphins showed higher binding affinity and sustained interactions with all three subsites of the ACE active site. An in vitro ACE inhibition assay showed that the IC50 of camel hemorphins were significantly lower than the IC50 of non-camel hemorphins. [ABSTRACT FROM AUTHOR]