INTRODUCTION
A fundamental characteristic of mammalian proteins and peptides is that they are composed predominantly of L-amino acids, with incorporation of D-amino acids occurring only rarely under physiological conditions (1–5). Recent advances in analytical chemistry and peptide-synthesis technologies have enabled systematic investigation and comparison of the properties of enantiomeric peptides, those containing the mirror-image configurations of amino acids (1, 5–9). For instance, the use of D-amino acid substitution offers an attractive strategy for modulating peptide and protein biological function, enhancing proteolytic stability, altering receptor affinity or signalling bias, and potentially improving therapeutic performance. Research in this area includes examples such as D-amino acid-containing polymer-peptide conjugates, which show markedly reduced immunogenicity compared to their L-counterparts (2−13).
Met-enkephalin is an ancient and evolutionarily conserved endogenous opioid pentapeptide with the amino acid sequence Tyr-Gly-Gly-Phe-Met (YGGFM) (14–17).
Its natural L-enantiomer (Fig. 1a) exhibits tissue and organ protective effects in different animal disease models, e.g., experimental allergic encephalomyelitis, histamine-induced bronchoconstriction, Arthus skin reaction, delayed skin reaction, adjuvant arthritis, cancer, allograft rejection, anaphylactic shock, and acetaminophen (APAP) induced hepatotoxicity (14–26). Mostly, the protective effects of L-Met-enkephalin are mediated via δ and ζ opioid receptors, and could be abolished with naltrexone, a competitive antagonist of the opioid receptors (15–17, 20, 21). The protective effects of L-Met-enkephalin could also be blocked with the antisense peptide IPPKY, which prevents its interaction with the opioid receptors (20, 25, 28).
Fig. 1. Structure of: a) L-Met-enkephalin; b) D-Met-enkephalin.
In contrast, the D-enantiomer structure of Met-enkephalin is characterized by D-amino acid substitution (chiral inversion) at the positions 1, 4 and 5 of the L-amino acid sequence (Fig. 1b). It is important to observe that amino acid glycine (G) at positions 2 and 3 is not a chiral amino acid, so this part of the molecule, including the glycine-glycine bond, is identical for both enantiomers (Fig. 1).
In this study we therefore investigated three key questions: (i) whether D-Met-enkephalin exhibits hepatoprotective activity in a model of acetaminophen-induced liver injury, (ii) whether its protective effect is modulated by naltrexone (to probe opioid receptor involvement) or by the antisense peptide IPPKY (to assess receptor-interaction specificity), and (iii) how its effect compares to the previously reported hepatoprotection of the L-enantiomer (20).
Met-enkephalin hepatoprotection has been examined previously only for the natural L-enantiomer in the same experimental model, where a 7.5 mg kg–1 dose produced the maximal effect (20). However, no biological or biophysical data exist for the D-enantiomer of Met-enkephalin, and the consequences of chiral inversion at positions 1, 4, and 5 on receptor-mediated hepatoprotection have not been addressed. The present work, therefore, represents a stereochemical extension of earlier studies: it examines whether D-Met-enkephalin retains hepatoprotective activity, whether receptor-mediated mechanisms are preserved, and whether its interaction with the cognate antisense peptide differs from that of the L-form. Answers to these questions are necessary to define the structure-function relationship between peptide chirality and opioid receptor-dependent cytoprotection.
EXPERIMENTAL
Treatment regimen and experimental model
The effects of D-Met-enkephalin were evaluated using the APAP-induced hepatotoxicity in male CBA mice. This murine model is useful for testing substances with potential hepatoprotective and anti-inflammatory effects (29, 30). The APAP reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) is responsible for damage to the centrilobular regions of the liver after overdose (30, 31). NAPQI reacts with sulfhydryl (thiol) groups of important intracellular proteins – particularly mitochondrial proteins involved in energy production, antioxidant defence-related proteins containing critical cysteine residues, and structural proteins that support cellular integrity-leading to mitochondrial dysfunction and cell death with the release of intracellular content (29). Damage-associated molecular patterns (DAMPs), e.g., nuclear protein HMGB1, nuclear DNA fragments, mitochondrial DNA, uric acid, ATP, and others, cause the transcriptional activation of pro-inflammatory cytokines in macrophages via DAMP receptors (29). Released proinflammatory mediators activate and recruit neutrophils and monocytes to the liver. Consequently, APAP overdose is accompanied by strong sterile inflammation of the liver (29). For clarity, all subsequent references to D-Met-enkephalin effects refer to our own experimental results.
Hepatotoxicity was induced following the procedure described by Guarner et al. with slight modifications (30–32). The experimental animals in the hepatotoxicity model were male CBA mice, 12–16 weeks old, weighing 20–25 g. The animals were bred at the Ruđer Bošković Institute and were kept under standard laboratory conditions (dark-light cycle (12/12 h), constant temperature (22 ± 1 °C) and humidity 55 %) with free access to water and standard food pellets (4 RF 21 GLP Mucedola srl, Italy). The experimental animals (n = 48) were randomly divided into five experimental groups (n = 8 animals per group), and a control group where animals were treated with saline (0.9 % NaCl).
For seven days, mice were given 300 mg L–1 phenobarbital (Phenobarbiton, PLIVA, Croatia) in drinking water. Prior to inducing liver damage by APAP, the animals were fasted overnight with free access to water. Acetaminophen (> 99 % purity; Sigma-Aldrich, USA) was given intragastrically (i.g.), in a dose of 150 mg kg–1 of body mass, dissolved in 0.9 % NaCl (37 °C) via a gastric tube, in a volume of 0.5 mL. Mice were re-fed after 4 h. Control animals were treated with physiological saline (0.9 % NaCl, Croatian Institute for Transfusion Medicine, Zagreb, Croatia).
The following test substances were given intraperitoneally (i.p.) 1 h before APAP administration, in a volume of 0.2 mL:
D-Met-enkephalin (Mr = 573.66, > 99 % purity; GenScript, USA);
Naltrexone hydrochloride (Mr = 377.86, > 99 % purity; Sigma-Aldrich;
Antisense peptides (IPPKY, Mr = 616.75, 98.5 % purity, and IPPKYW, Mr = 802.96, > 99 % purity, GenScript, USA);
Administration of naltrexone and D-Met-enkephalin (naltrexone was given 30 min prior to D-Met-enkephalin);
Mixture of D-Met-enkephalin and antisense peptide IPPKY (mixed together 30 minutes prior to the administration).
Twenty-four hours after APAP administration, the experimental animals were sacrificed. Fifteen minutes before sacrifice, 250 IU heparin was given i.p. to each animal, and trunk blood was collected into heparinised tubes. Plasma was separated by centrifugation for 5 minutes at 8000 g and was stored at –20 °C for 24 h before transaminase activity determination. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity were determined from plasma on an Olympus AU® 400 analyser using standard reagents.
Sections of the liver were fixed in 10 % phosphate-buffered formalin and embedded in paraffin for histopathological analysis. Three specimens of the liver tissue were analysed. Each specimen was cut into six sections of 3 μm and stained with hemalum-eosin. Liver lesions were graded using light microscope (×100) on 0–5 scale: 0 = no lesions; 1 = minimal lesions (individual necrotic cells); 2 = mild lesions (10 to 25 % of necrotic cells or mild diffuse degenerative changes); 3 = moderate lesions (25 to 40 % of necrotic cells); 4 = marked lesions (40 to 50 % of necrotic cells); and 5 = severe lesions (> 50 % of necrotic cells) (33). The final score for each liver was the consensus score of all examined sections.
All the experiments were performed according to theethical guidelines of the International Council for Laboratory Animal Science (ICLAS), Council Directive 2010/63/EU, and Croatian Animal Protection Act (Official Gazette 135/06). All animal experimentation protocols were approved by the Ruđer Bošković Institute and by the Croatian Ministry of Agriculture (ethics approval: No. 525-10/0255-13-2).
Circular dichroism spectroscopy
The circular dichroism (CD) spectra of L- and D-Met-enkephalin enantiomers were acquired at room temperature with a Jasco J-815 CD spectropolarimeter (Jasco Inc., USA), in a 0.2 mm optical path quartz precision cell. The peptides were dissolved in 10 mmol L–1 phosphate buffer at pH = 7.4 and 25 °C. Concentration of the peptides was 2 mg mL–1. Circular dichroism spectra show a random coil structure of both Met-enkephalin peptides, and a typical mirror image CD spectra of L- and D-enantiomers due to L/D isomerisation (Fig. 2).
Fig. 2. Circular dichroism spectroscopy analysis of L- and D-Met-enkephalin structures.
Peptide binding assay using tryptophan fluorescence spectroscopy
The binding of D-Met-enkephalin and its antisense peptide ligand, modified by the C-terminal tryptophan fluorophore (IPPKYW), was measured by OLIS RSM 1000F spectrofluorometer (Olis, Inc., USA) equipped with thermostated cell at 25 °C (34, 35). Both reactants (D-Met-enkephalin and IPPKYW) were fluorophores, and the third spectrally active species was attributed to the complex of two reactants. D-phenylalanine, present in D-Met-enkephalin, which was in excess, had a much smaller quantum yield than the tryptophan present in IPPKYW (21). The excitation wavelength at 290 nm was chosen to diminish the fluorescence of D-phenylalanine and maximise the fluorescence of antisense tryptophan (Fig. 3) (20).
Fig. 3. Titration of 2.5 μmol L–1 solution of IPPKYW with D-Met-enkephalin at 25 °C and pH = 7.4 in 10 mmol L–1 phosphate buffer. The concentration of D-Met-enkephalin varied from 2.5 to 500 μmol L–1. Fluorescence in arbitrary units (AU) is given as a ratio of signals obtained from sample and reference PMTs. Inset: Fitting curve at 350 nm.
SPECFIT software was used to analyse all spectra in fluorescence titrations (Fig. 3). Three spectrally active species were suggested by single value decomposition (SVD) statistical analysis (20, 35–37). Data analysis suggested 1 to 1 complex formation and did not indicate the presence of higher-order complexes. Dissociation constant of the complex (Kd = 3.7 ± 0.9 μmol L–1) was calculated according to the model given by Equation 1 and Equation 2:
IPPKYW-D-YGGFM ↔ IPPKYW + D-YGGFM (1)
(2)
GraphPad Prism for Windows (version 10) and KyPlot (version 6) were used for data plotting and statistical analysis. Descriptive statistics for AST and ALT activities were based on means, medians, and standard deviations. Minimum, first quartile (Q1), median, third quartile (Q3) and maximum were used to describe liver necrosis scores. Data shown in plots represent medians and interquartile ranges. Differences between the groups were analysed using Steel’s test, a non-parametric multiple comparison test that compares treatment groups with control. All applied tests were two-tailed, and the critical p-value for accepting or rejecting the null hypothesis was 0.05.
RESULTS AND DISCUSSION
Hepatoprotective effects of D-Met-enkephalin
The hepatoprotective effects of D-Met-enkephalin were assessed using two standard criteria: (i) plasma activities of the liver enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and (ii) the histopathological necrosis score (scale 0–5). D-Met-enkephalin produced dose-dependent hepatoprotective effects in the acetaminophen-induced liver injury model, as reflected by plasma AST and ALT activities and histopathological necrosis score (Figs. 4–6). Control animals treated with 0.9 % NaCl showed markedly elevated AST (mean 7500 U L–1) and ALT (mean 3606 U L–1) levels and high necrosis scores (median 3.5), indicating severe liver injury. Treatment with D-Met-enkephalin at 0.5, 5, and 20 mg kg–1 significantly reduced AST (means 486.3, 285.0, and 430.7 U L–1; p = 0.0065), ALT (means 458.4, 158.3, and 196.6 U L–1; p = 0.0065), and necrosis scores (medians 2.0, 1.5, and 2.0; p = 0.0151, 0.0115, and 0.0265, respectively), with maximal protection observed at 5 mg kg–1. Although this optimal dose is lower than the 7.5 mg kg–1 reported for the natural L-enantiomer (mean ALT = 794 U L–1, p = 0.0072; AST = 1313 U L–1, p = 0.0039 and median necrosis score = 2.5, p = 0.0094) (20), both values fall within a similar effective range. The difference may reflect enantiomer-specific pharmacodynamic properties as well as methodological differences among studies. These doses are also consistent with the optimal protective range (4–10 mg kg–1) described in other models of inflammatory and autoimmune disease in mice, rats, and guinea pigs (21, 23–26). The highest dose of D-Met-enkephalin (50 mg kg–1), did not confer hepatoprotection, as AST (mean 11,884 U L–1; p = 0.262), ALT (mean 5406 U L–1; p = 0.678), and necrosis scores (median 4.0; p = 0.446) were comparable to control values (Fig. 4–6).
Similarly to the results for L-enantiomer (20), the effects of D-Met-enkephalin in the model of acetaminophen-induced liver toxicity were also characterised by a U-shaped curve relationship between the applied doses and observed protective effects. This was valid for all three parameters relevant for this experimental model – AST, ALT, and the liver necrosis score (Fig. 4–6). The U-shaped curve is often observed with peptide ligands, including opioid system ligands characterised by low-dose stimulation and high-dose inhibition (20, 41).
Fig. 4. Dose-dependent effects of D-Met-enkephalin on plasma AST activity 24 h after acetaminophen administration. Data are shown as medians and interquartile ranges.
Fig. 5. Dose-dependent effects of D-Met-enkephalin on plasma ALT activity 24 h after acetaminophen administration. Data are shown as medians and interquartile ranges.
Fig. 6. Dose-dependent effects of D-Met-enkephalin on liver necrosis (scale 0–5) 24 h after acetaminophen administration. Data are shown as medians and interquartile ranges.
Interestingly, D-Met-enkephalin doses in the range of 0.5–5 mg kg–1 have somewhat better protective potency than the similar range of L-enantiomer doses (20). The most probable explanation is that the substitution of L-amino acid with D-amino acid at positions 1, 4, and 5 influences the enzymatic breakdown of the molecule, prolonging its life and consequently enhancing its effects (3, 42). However, additional studies of the D-Met-enkephalin degradation with specific enzymes, e.g., aminopeptidase N, enkephalinase A, enkephalinase B and carboxypeptidase A6 are needed (Fig. 7).
Fig. 7. Enzymes involved in the breakdown of the Met-enkephalin molecule.
To date, no published data have described the biological effects of the D-Met-enkephalin enantiomer. In this study, we demonstrated for the first time that D-Met-enkephalin exhibits hepatoprotective activity in a model of acetaminophen (APAP)-induced liver injury, a well-established system for screening compounds with hepatoprotective and anti-inflammatory properties (7, 20, 31, 33, 38). This model is particularly suitable for evaluating pharmacologically active agents with anti-inflammatory and anti-necrotic potential (28, 39–40).
Modulation of D-Met-enkephalin hepatoprotection with receptor and peptide blockade
The effects of L-Met-enkephalin are mediated via δ and ζ opioid receptors and could be abolished by the opioid receptor antagonist naltrexone (15, 17). Martinić et al. showed that naltrexone, a competitive and long-acting antagonist of the opioid receptors, blocks the hepatoprotective effects of L-Met-enkephalin (20). The same phenomenon was observed with L-Met-enkephalin antisense peptide (IPPKY) (20). Consequently, we investigated D-Met-enkephalin hepatoprotection from the standpoint of the opioid receptor blockade (with naltrexone) and D-Met-enkephalin blockade (with antisense peptide IPPKY).
The effects of opioid receptor blockade with naltrexone were investigated in group 2 and group 3 (Table I). The administration of naltrexone 30 minutes prior to D-Met-enkephalin completely abolished its protective effects. The mortality rate of animals due to acetaminophen toxicity in the groups treated with naltrexone (6/8, 75 %) and naltrexone + D-Met-enkephalin (5/8, 62.5 %) was high. This high mortality rate probably results from the blockade of both pharmacologically applied D-Met-enkephalin, as well as the blockade of endogenous L-Met-enkephalin (20). Similar mortality was observed when antisense peptide was applied alone (5/8, 62.5 %) or together with D-Met-enkephalin (6/8, 75 %). Sense-antisense peptide complex, naltrexone and antisense peptide could also contribute to the hepatotoxicity, but this could not be evaluated due to the high number of deceased animals (Table I).
Table I. Mortality in different experimental groups when the D-Met-enkephalin effects were blocked with naltrexone and antisense peptide
The blockade of the best protective dose of D-Met-enkephalin (5 mg kg–1) with naltrexone suggests that hepatoprotection induced by D-enantiomer is mediated via opioid receptors, similarly to the situation with L-enantiomer. In addition, the blockade of D-Met-enkephalin with antisense peptide allowed us to observe its biological effect in the state of the preserved receptor function, enabling other endogenous substances to act concomitantly (31).
The effects of Met-enkephalin blockade with an antisense peptide were investigated in groups 4 and 5 (Table I). A mixture of D-Met-enkephalin and antisense peptide (mixed together 30 minutes prior to the administration) blocked the hepatoprotective effects of D-Met-enkephalin and confirmed the results of the in vitro binding assay (Fig. 3).
It is known that D-amino acid substitution in the peptide sequence may improve its binding and preserve function and/or structure. Dissociation constant for the complexes of D-Met-enkephalin with antisense peptide was 3.7 ± 0.9 μmol L–1 (mean ± SD). Martinić et al. showed that the dissociation constant for the complex of L-Met-enkephalin with antisense peptide was 19 ± 3 μmol L–1 (mean ± SD) (20). Those results indicate a slightly higher affinity of the D-enantiomer for the antisense peptide. It also supports the concept of the sense-antisense peptide interaction, stating that antisense peptides specified by the complementary RNAs bind to sense peptides, with enhanced specificity and affinity (27, 33, 34, 43, 44, 46). In this way, antisense peptides could abolish the biological activity of the peptide hormones, a fact experimentally verified for more than 40 different ligand-receptor systems (20, 27, 33, 34, 43, 44, 46).
Preclinical and early-phase clinical trials of L-Met-enkephalin, also considered to be an opioid growth factor (OGF) (17) and cytokine (15), showed that its administration is safe and non-toxic (14–19). The obtained data suggest that both L- and D-Met-enkephalin enantiomers might possess therapeutic potential in inflammatory diseases of the liver and in acetaminophen overdose. Although these results are limited to an experimental model, the hepatoprotective activity of D-Met-enkephalin suggests that D-amino-acid-modified enkephalins may merit further preclinical investigation. Additional studies on pharmacokinetics, safety, and the mechanism of action will be needed before any potential clinical application can be considered.
CONCLUSIONS
The earlier study on L-Met-enkephalin demonstrated receptor-dependent hepatoprotection but did not address how stereochemical inversion influences biological activity, receptor engagement, or antisense peptide recognition. Although the same APAP-induced hepatotoxicity model was used in our earlier study of the L-enantiomer, the present study demonstrates that the D-enantiomer of Met-enkephalin possesses distinct yet comparable hepatoprotective activity to its natural L-form in acetaminophen-induced hepatotoxicity in male CBA mice. D-Met-enkephalin exhibited dose-dependent protection within the 0.5–20 mg kg–1 range, with maximal efficacy at 5.0 mg kg–1. The observed effects were both peptide- and receptor-specific, mediated via opioid receptor activation and abolished by the opioid antagonist naltrexone. These findings indicate that partial chiral inversion does not abolish the biological activity of Met-enkephalin, likely owing to the presence of achiral glycine residues preserving the peptide’s functional conformation. The results further suggest that strategic incorporation of D-amino acids can yield bioactive peptides with retained receptor affinity and improved stability, offering new perspectives for the rational design of peptide-based hepatoprotective and cytoprotective therapeutics.
Acknowledgment. – The support of the Croatian Ministry of Science and Education is gratefully acknowledged (grant No. 098-0982929-2524). The authors thank Paško Konjevoda, PhD, Ruđer Bošković Institute, Zagreb, Croatia, and Prof. Mario Gabričević, University of Zagreb Faculty of Pharmacy and Biochemistry, Zagreb, Croatia, for advice on experimental design and statistical analysis.
Conflict of interest. – The authors declare that they have no conflicts of interest.
Funding. – The support of the Croatian Ministry of Science, Education and Sports (grant No. 098-0982929-2524) is thankfully appreciated.
Authors contributions. – Conceptualisation, P.T. and N.Š.; design of D-Met-enkephalin enantiomer and antisense peptides, N.Š. and R.M., investigation, P.T., R.S., A.B.-B., and T.W.; statistical analyses, M.M. and P.T.; spectroscopic studies and transaminase measurement, T.W., M.M., and P.T.; histopathology analysis and scoring, M.K. All authors have read and agreed to the published version of the manuscript.