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

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

In Vitro Functional Profiling of Fentanyl and Nitazene Analogs at the µ-Opioid Receptor Reveals High Efficacy for Gi Protein Signaling.

Novel synthetic opioids (NSOs), including both fentanyl and non-fentanyl analogs that act as µ-opioid receptor (MOR) agonists, are associated with serious intoxication and fatal overdose. Previous studies proposed that G-protein-biased MOR agonists are safer pain medications, while other evidence indicates that low intrinsic efficacy at MOR better explains the reduced opioid side effects. Here, we characterized the in vitro functional profiles of various NSOs at the MOR using adenylate cyclase inhibition and ß-arrestin2 recruitment assays, in conjunction with the application of the receptor depletion approach. By fitting the concentration-response data to the operational model of agonism, we deduced the intrinsic efficacy and affinity for each opioid in the Gi protein signaling and ß-arrestin2 recruitment pathways. Compared to the reference agonist [d-Ala2,N-MePhe4,Gly-ol5]enkephalin, we found that several fentanyl analogs were more efficacious at inhibiting cAMP production, whereas all fentanyl analogs were less efficacious at recruiting ß-arrestin2. In contrast, the non-fentanyl 2-benzylbenzimidazole (i.e., nitazene) analogs were highly efficacious and potent in both the cAMP and ß-arrestin2 assays. Our findings suggest that the high intrinsic efficacy of the NSOs in Gi protein signaling is a common property that may underlie their high risk of intoxication and overdose, highlighting the limitation of using in vitro functional bias to predict the adverse effects of opioids. In addition, the extremely high potency of many NSOs now infiltrating illicit drug markets further contributes to the danger posed to public health.

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

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

Impact of angiotensin receptor-neprilysin inhibition (ARNI) in improving ejection fraction and left and right ventricular remodeling in heart failure.

Angiotensin receptor neprilysin inhibitors (ARNI), a new therapeutic class of agents acting on the renin angiotensin aldosterone system (RAAS) and neutral endopeptidase system has been developed in treatment of ventricular remodeling and has attracted considerable attention. The first in class is LCZ696, which is a molecule that combines Valsartan (ARB) and Sacubitril (neprilysin inhibitor) within a single substance. Sacubitril-Valsartan is the first angiotensin receptor enkephalin inhibitors (ARNI), which can block angiotensin II type 1 receptor (AT1R) while inhibiting enkephalin (NEP) and effectively reverse ventricular remodeling in heart failure patients. It has been recommended by the European and American authoritative guidelines on heart failure as Class I for the treatment of chronic heart failure particularly as intensive care medicine. Sacubitril-Valsartan demonstrated significant effects in improving left ventricular performance and remodeling in patients with heart failure with reduced ejection fraction. Sacubitril acts on increased levels of circulating natriuretic peptides by preventing their enzymatic breakdown and Valsartan, which acts to lessen the effects of the RAAS. However, not more research has been done on its effects on the right ventricle remodeling. This review aimed to assess the impact of angiotensin receptor neprilysin inhibitors on left and right ventricular remodeling in heart failure patients.

Plasma proenkephalin A and incident chronic kidney disease and albuminuria in the REasons for Geographic And Racial Differences in Stroke (REGARDS) cohort.

BACKGROUND: Plasma proenkephalin A (PENK-A) is a precursor of active enkephalins. Higher blood concentrations have been associated with estimated glomerular filtration rate (eGFR) decline in European populations. Due to the significant disparity in incident chronic kidney disease (CKD) between White and Black people, we evaluated the association of PENK-A with incident CKD and other kidney outcomes among a biracial cohort in the U.S. METHODS: In a nested cohort of 4,400 participants among the REasons for Geographic And Racial Differences in Stroke, we determined the association between baseline PENK-A concentration and incident CKD using the creatinine-cystatin C CKD-EPI 2021 equation without race coefficient, significant eGFR decline, and incident albuminuria between baseline and a follow-up visit 9.4 years later. We tested for race and sex interactions. We used inverse probability sampling weights to account for the sampling design. RESULTS: At baseline, mean (SD) age was 64 (8) years, 49% were women, and 52% were Black participants. 8.5% developed CKD, 21% experienced ≥ 30% decline in eGFR and 18% developed albuminuria. There was no association between PENK-A and incident CKD and no difference by race or sex. However, higher PENK-A was associated with increased odds of progressive eGFR decline (OR: 1.12; 95% CI 1.00, 1.25). Higher PENK-A concentration was strongly associated with incident albuminuria among patients without diabetes mellitus (OR: 1.29; 95% CI 1.09, 1.53). CONCLUSION: While PENK-A was not associated with incident CKD, its associations with progression of CKD and incident albuminuria, among patients without diabetes, suggest that it might be a useful tool in the evaluation of kidney disease among White and Black patients.

Proenkephalin deletion in hematopoietic cells induces intestinal barrier failure resulting in clinical feature similarities with irritable bowel syndrome in mice.

Opioid-dependent immune-mediated analgesic effects have been broadly reported upon inflammation. In preclinical mouse models of intestinal inflammatory diseases, the local release of enkephalins (endogenous opioids) by colitogenic T lymphocytes alleviate inflammation-induced pain by down-modulating gut-innervating nociceptor activation in periphery. In this study, we wondered whether this immune cell-derived enkephalin-mediated regulation of the nociceptor activity also operates under steady state conditions. Here, we show that chimeric mice engrafted with enkephalin-deficient bone marrow cells exhibit not only visceral hypersensitivity but also an increase in both epithelial paracellular and transcellular permeability, an alteration of the microbial topography resulting in increased bacteria-epithelium interactions and a higher frequency of IgA-producing plasma cells in Peyer's patches. All these alterations of the intestinal homeostasis are associated with an anxiety-like behavior despite the absence of an overt inflammation as observed in patients with irritable bowel syndrome. Thus, our results show that immune cell-derived enkephalins play a pivotal role in maintaining gut homeostasis and normal behavior in mice. Because a defect in the mucosal opioid system remarkably mimics some major clinical symptoms of the irritable bowel syndrome, its identification might help to stratify subgroups of patients.

Lateral hypothalamic proenkephalin neurons drive threat-induced overeating associated with a negative emotional state.

Psychological stressors, like the nearby presence of a predator, can be strong enough to induce physiological/hormonal alterations, leading to appetite changes. However, little is known about how threats can alter feeding-related hypothalamic circuit functions. Here, we found that proenkephalin (Penk)-expressing lateral hypothalamic (LHPenk) neurons of mice exposed to predator scent stimulus (PSS) show sensitized responses to high-fat diet (HFD) eating, whereas silencing of the same neurons normalizes PSS-induced HFD overconsumption associated with a negative emotional state. Downregulation of endogenous enkephalin peptides in the LH is crucial for inhibiting the neuronal and behavioral changes developed after PSS exposure. Furthermore, elevated corticosterone after PSS contributes to enhance the reactivity of glucocorticoid receptor (GR)-containing LHPenk neurons to HFD, whereas pharmacological inhibition of GR in the LH suppresses PSS-induced maladaptive behavioral responses. We have thus identified the LHPenk neurons as a critical component in the threat-induced neuronal adaptation that leads to emotional overconsumption.

Endogenous opioid system modulates conditioned cocaine reward in a sex-dependent manner.

Cocaine predictive cues and contexts exert powerful control over behaviour and can incite cocaine seeking and taking. This type of conditioned behaviour is encoded within striatal circuits, and these circuits and behaviours are, in part, regulated by opioid peptides and receptors expressed in striatal medium spiny neurons. We previously showed that augmenting levels of the opioid peptide enkephalin in the striatum facilitates acquisition of cocaine conditioned place preference (CPP), while opioid receptor antagonists attenuate expression of cocaine CPP. However, whether striatal enkephalin is necessary for acquisition of cocaine CPP and maintenance during extinction remains unknown. To address this, we generated mice with a targeted deletion of enkephalin from dopamine D2-receptor expressing medium spiny neurons and tested them in a cocaine CPP paradigm. Low striatal enkephalin levels did not attenuate acquisition of CPP. However, expression of preference, assessed after acute administration of the opioid receptor antagonist naloxone, was blocked in females, regardless of genotype. When saline was paired with the cocaine context during extinction sessions, females, regardless of genotype, extinguished preference faster than males, and this was prevented by naloxone when paired with the cocaine context. We conclude that while striatal enkephalin is not necessary for acquisition, expression, or extinction of cocaine CPP, expression and extinction of cocaine preference in females is mediated by an opioid peptide other than striatal enkephalin. The unique sensitivity of females to opioid antagonists suggests sex should be a consideration when using these compounds in the treatment of cocaine use disorder.

Nav1.7 is essential for nociceptor action potentials in the mouse in a manner independent of endogenous opioids.

Loss-of-function mutations in Nav1.7, a voltage-gated sodium channel, cause congenital insensitivity to pain (CIP) in humans, demonstrating that Nav1.7 is essential for the perception of pain. However, the mechanism by which loss of Nav1.7 results in insensitivity to pain is not entirely clear. It has been suggested that loss of Nav1.7 induces overexpression of enkephalin, an endogenous opioid receptor agonist, leading to opioid-dependent analgesia. Using behavioral pharmacology and single-cell RNA-seq analysis, we find that overexpression of enkephalin occurs only in cLTMR neurons, a subclass of sensory neurons involved in low-threshold touch detection, and that this overexpression does not play a role in the analgesia observed following genetic removal of Nav1.7. Furthermore, we demonstrate using laser speckle contrast imaging (LSCI) and in vivo electrophysiology that Nav1.7 function is required for the initiation of C-fiber action potentials (APs), which explains the observed insensitivity to pain following genetic removal or inhibition of Nav1.7.

Efficacy of dual enkephalinase inhibition in a preclinical migraine model is mediated by activation of peripheral delta opioid receptors.

OBJECTIVE: The aim of this study was to evaluate whether elevating levels of enkephalin by inhibiting their degradation can attenuate stress-induced migraine-like behaviors in mice. BACKGROUND: Previous studies in animals have suggested the delta opioid receptor (DOR) as a novel migraine target. The primary endogenous ligands for DOR are enkephalins and their levels can be increased by pharmacological inhibition of enkephalinases; however, it is not clear whether enkephalinase inhibition can be efficacious in preclinical migraine models through activation of DOR or whether other opioid receptors might be involved. Further, it is not clear whether opioid receptors in the central nervous system are necessary for these effects. METHODS: This study used a model of repetitive restraint stress in mice that induces periorbital hypersensitivity and priming to the nitric oxide donor sodium nitroprusside (SNP; 0.1 mg/kg). Von Frey filaments were used to measure periorbital mechanical thresholds and grimace scores were evaluated by observing mouse facial features. Animals were treated with the dual enkephalinase inhibitor (DENKI) PL37. RESULTS: On day two post-stress, PL37 given to mice via either intravenous injection (10 mg/kg) or oral gavage (20 mg/kg) significantly attenuated stress-induced periorbital hypersensitivity and facial grimace responses. Additionally, both intravenous (10 mg/kg) and oral gavage (20 mg/kg) of PL37 prior to SNP (0.1 mg/kg) administration on day 14 post-stress significantly reduced SNP-induced facial hypersensitivity. Injection of the DOR antagonist naltrindole (0.1 mg/kg) but not the mu-opioid receptor antagonist CTAP (1 mg/kg) prior to PL37 treatment blocked the effects. Finally, pretreatment of mice with the peripherally restricted opioid receptor antagonist naloxone methiodide (5 mg/kg) blocked the effects of PL37. CONCLUSIONS: These data demonstrate that inhibiting enkephalinases, and thus protecting enkephalins from degradation, attenuates stress-induced migraine-like behavior via activation of peripheral DOR. Peripheral targeting of endogenous opioid signaling may be an effective therapeutic strategy for migraine.

Prolonged Dystocic Labor in Neuraxial Analgesia and the Role of Enkephalin Neurotransmitters: An Experimental Study.

The investigation studied the enkephalinergic neuro fibers (En) contained in the Lower Uterine Segment (LUS) during the prolonged dystocic labor (PDL) with Labor Neuraxial Analgesia (LNA). PDL is generally caused by fetal head malpositions in the Occiput Posterior Position (OPP), Persistent Occiput Posterior Position (POPP), in a transverse position (OTP), and asynclitism (A), and it is detected by Intrapartum Ultrasonography (IU). The En were detected in the LUS samples picked up during cesarean section (CS) of 38 patients undergoing urgent CS in PDL, compared to 37 patients submitted to elective CS. Results were statistically evaluated to understand the differences in En morphological analysis by scanning electron microscopy (SEM) and by fluorescence microscopy (FM). The LUS samples analysis showed an important reduction in En in LUS of CS for the PDL group, in comparison with the elective CS group. The LUS overdistension, by fetal head malpositions (OPP, OTP, A) and malrotations, lead to dystocia, modification of vascularization, and En reduction. The En reduction in PDL suggests that drugs used during the LNA, usually local anesthetics and opioids, cannot control the "dystocic pain", that differs from normal labor pain. The IU administration in labor and the consequent diagnosis of dystocia suggest stopping the numerous and ineffective top-up drug administration during LNA, and to shift the labor to operative vaginal delivery or CS.

Influence of Mutations of Conserved Arginines on Neuropeptide Binding in the DPP III Active Site.

Dipeptidyl peptidase III (DPP III), a zinc exopeptidase, is involved in the final steps of intercellular protein degradation and has a marked affinity for opioid peptides such as enkephalins and endomorphins. Recently, we characterized a number of neuropeptides as potential substrates and inhibitors of human DPP III and provided an explanation for their differential behavior. These studies prompted us to investigate the influence of the conserved R399 and R669 on neuropeptides binding to DPP III. Measuring kinetic parameters in inhibitory assays, we found that mutation of R669 to Ala or Met significantly reduced the inhibitory properties of the slow substrates tynorphin and valorphin, whereas the effects on binding of the good substrates Arg2-2NA and Leu-enkephalin were small. Molecular dynamics simulations of wild-type (WT) and mutant DPP III complexes with Leu-enkephalin, tynorphin, valorphin, and Arg2-2NA in conjunction with calculations of binding free energies revealed that the lower inhibitory potency of slow substrates in the R669A mutant can be explained by the lower binding affinity of tynorphin and the higher propensity of valorphin to hydrolyze in the mutant than in WT. The R399A mutation was shown to affect the binding and/or hydrolysis of both good and slow substrates, with the effects on Leu-enkephalin being the most pronounced.

Enkephalin-δ Opioid Receptor Signaling Mediates Glucoprivic Suppression of LH Pulse and Gluconeogenesis in Female Rats.

Energy availability is an important regulator of reproductive function at various reproductive phases in mammals. Glucoprivation induced by 2-deoxy-D-glucose (2DG), an inhibitor of glucose utilization, as an experimental model of malnutrition suppresses the pulsatile release of GnRH/LH and induces gluconeogenesis. The present study was performed with the aim of examining whether enkephalin-δ-opioid receptor (DOR) signaling mediates the suppression of pulsatile GnRH/LH release and gluconeogenesis during malnutrition. The administration of naltrindole hydrochloride (NTI), a selective DOR antagonist, into the third ventricle blocked the suppression of LH pulses and part of gluconeogenesis induced by IV 2DG administration in ovariectomized rats treated with a negative feedback level of estradiol-17â€…ß (OVX + low E2). The IV 2DG administration significantly increased the number of Penk (enkephalin gene)-positive cells coexpressing fos (neuronal activation marker gene) in the paraventricular nucleus (PVN), but not in the arcuate nucleus (ARC) in OVX + low E2 rats. Furthermore, double in situ hybridization for Penk/Pdyn (dynorphin gene) in the PVN revealed that approximately 35% of the PVN Penk-expressing cells coexpressed Pdyn. Double in situ hybridization for Penk/Crh (corticotropin-releasing hormone gene) in the PVN and Penk/Kiss1 (kisspeptin gene) in the ARC revealed that few Penk-expressing cells coexpressed Crh and Kiss1. Taken together, these results suggest that central enkephalin-DOR signaling mediates the suppression of pulsatile LH release during malnutrition. Moreover, the current study suggests that central enkephalin-DOR signaling is also involved in gluconeogenesis during malnutrition in female rats.

The inhibition of enkephalin catabolism by dual enkephalinase inhibitor: A novel possible therapeutic approach for opioid use disorders.

Despite the increasing impact of opioid use disorders on society, there is a disturbing lack of effective medications for their clinical management. An interesting innovative strategy to treat these disorders consists in the protection of endogenous opioid peptides to activate opioid receptors, avoiding the classical opioid-like side effects. Dual enkephalinase inhibitors (DENKIs) physiologically activate the endogenous opioid system by inhibiting the enzymes responsible for the breakdown of enkephalins, protecting endogenous enkephalins and increasing their half-lives and physiological actions. The activation of opioid receptors by the increased enkephalin levels, and their well-demonstrated safety, suggests that DENKIs could represent a novel analgesic therapy and a possible effective treatment for acute opioid withdrawal, as well as a promising alternative to opioid substitution therapy minimizing side effects. This new pharmacological class of compounds could bring effective and safe medications avoiding the major limitations of exogenous opioids, representing a novel approach to overcome the problem of opioid use disorders. LINKED ARTICLES: This article is part of a themed issue on Advances in Opioid Pharmacology at the Time of the Opioid Epidemic. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v180.7/issuetoc.

Proenkephalin A as a marker for glomerular filtration rate in critically ill children: validation against gold standard iohexol GFR measurements.

OBJECTIVES: Accurate determination of glomerular filtration rate (GFR) is important. Several endogenous biomarkers exist for estimating GFR, yet, they have limited accuracy, especially in the paediatric population. Proenkephalin A 119-159 (PENK) is a novel and promising GFR marker, but its relation with age in children remains unknown. Also, the value of PENK has never been validated against measured GFR (mGFR) in children when compared to traditional GFR markers including serum creatinine (SCr), SCr-based estimated GFR (eGFR) and cystatin C (cysC). METHODS: Critically ill children and term-born neonates were included in this single-centre, prospective study. Iohexol-based mGFR, SCr, and cysC were determined in each patient. eGFR was calculated using the bedside Schwartz equation, incorporating SCr and height. Spearman correlation coefficients were calculated to determine the correlation between mGFR and PENK, SCr, cysC and eGFR. RESULTS: For 97 patients (56 children and 41 neonates), mGFR, SCr, cysC and PENK levels were available. PENK levels were higher in young children and decreased to adult PENK reference values around two years of age. PENK levels were highly correlated with mGFR (ρ=-0.88, p<0.001), and similar to mGFR-eGFR correlation (ρ=-0.87, p<0.001). For cysC and SCr the correlation with mGFR was lower (ρ=-0.77 and ρ=-0.46, respectively. Both p<0.001). CONCLUSIONS: The correlation of PENK with mGFR was as good as SCr-based eGFR-mGFR correlation. To determine the added value of PENK in paediatric clinical care and prior to implementation, PENK reference values are needed and the development and validation of a paediatric PENK-based eGFR equation is necessary.

NaV1.7 Channel Blocker [Ala5, Phe6, Leu26, Arg28]GpTx-1 Attenuates CFA-induced Inflammatory Hypersensitivity in Rats via Endogenous Enkephalin Mechanism.

Venom-derived NaV1.7 channel blockers have promising prospects in pain management. The 34-residue tarantula peptide GpTx-1 is a potent NaV1.7 channel blocker. Its powerful analog [Ala5, Phe6, Leu26, Arg28]GpTx-1 (GpTx-1-71) displayed excellent NaV1.7 selectivity and analgesic properties in mice. The current study aimed to elucidate the anti-hyperalgesic activities of GpTx-1-71 in inflammatory pain and reveal the underlying mechanisms. Our results demonstrated that intrathecal and intraplantar injections of GpTx-1-71 dose-dependently attenuated CFA-induced inflammatory hypersensitivity in rats. Moreover, GpTx-1-71-induced anti-hyperalgesia was significantly reduced by opioid receptor antagonists and the enkephalin antibody and diminished in proenkephalin (Penk) gene knockout animals. Consistently, GpTx-1-71 treatment increased the enkephalin level in the spinal dorsal horn and promoted the Penk transcription and enkephalin release in primary dorsal root ganglion (DRG) neurons, wherein sodium played a crucial role in these processes. Mass spectrometry analysis revealed that GpTx-1-71 mainly promoted the secretion of Met-enkephalin but not Leu-enkephalin from DRG neurons. In addition, the combination of subtherapeutic Met-enkephalin and GpTx-1-71 produced synergistic anti-hyperalgesia in CFA-induced inflammatory hypersensitivity. These findings suggest that the endogenous enkephalin pathway is essential for GpTx-1-71-induced spinal and peripheral analgesia in inflammatory pain. PERSPECTIVE: This article presents a possible pharmacological mechanism underlying NaV1.7 blocker-induced analgesia in inflammatory pain, which helps us to better understand and develop venom-based painkillers for incurable pain.

Utility of Measuring Circulating Bio-Adrenomedullin and Proenkephalin for 30-Day Mortality Risk Prediction in Patients with COVID-19 and Non-COVID-19 Interstitial Pneumonia in the Emergency Department.

Background and Objectives: In order to accelerate the risk stratification of patients referred to the Emergency Department (ED) with interstitial pneumonia, it could be useful to provide new and effective laboratory tests for use. The aim of our study was to evaluate the prognostic role of two biomarkers, bio-adrenomedullin (Bio-ADM) and proenkephalin (penKid), in patients with interstitial pneumonia (IP) at ED admission. Materials and Methods: In 153 consecutive patients with IP, both from COVID-19 or non-COVID-19 etiology, we measured, in a prospective observational manner, penKid and Bio-ADM at ED admission and after 24 h. In order to evaluate patient outcomes, 30-day follow-ups were also performed. The endpoints were 24 h, 10-day, and 30-day mortality. Results: Both biomarkers were shown to be good predictors of adverse events at 30 days, with Bio-ADM outperforming penKid. Bio-ADM was linked with 24 h and 10-day patient mortality. Moreover, PenKid was related to parameters defining worsening kidney function. Conclusions: Both in patients with COVID-19 or non-COVID-19 interstitial pneumonia at ED admission, Bio-ADM and penKid were good predictors of patient mortality. To evaluate these two biomarkers could be considered to be useful during the first evaluation in the ED when integrated with clinical scores.

N-Terminally Lipidated Sialorphin Analogs-Synthesis, Molecular Modeling, In Vitro Effect on Enkephalins Degradation by NEP and Treatment of Intestinal Inflammation in Mice.

Pharmacotherapy for inflammatory bowel disease (IBD) is difficult, and some patients do not respond to currently available treatments. Therefore, the discovery of novel anti-IBD agents is imperative. Our aim was the synthesis of lipidated analogs of sialorphin and the in vitro characterization of their effect on the degradation of Met-enkephalin by neutral endopeptidase (NEP). We also investigated in vivo whether the most active inhibitor (peptide VIII) selected in the in vitro studies could be a potential candidate for the treatment of colitis. Peptides were synthesized by the solid-phase method. Molecular modeling technique was used to explain the effect of fatty acid chain length in sialorphin analogs on the ligand-enzyme interactions. The anti-inflammatory effect was evaluated in the dextran sulphate sodium (DSS)-induced model of colitis in mice. Peptide VIII containing stearic acid turned out to be in vitro the strongest inhibitor of NEP. We have also shown that the length of the chain of stearic acid fits the size of the grove of NEP. Peptides VII and VIII exhibited in vivo similar anti-inflammatory activity. Our results suggest that lipidation of sialorphin molecule is a promising direction in the search for NEP inhibitors that protect enkephalins.

Molecular insights into the mechanism of sugar-modified enkephalin binding to opioid receptors.

Opioid receptors (delta, kappa, and mu) belong to the G protein-coupled receptor (GPCR) superfamily. They are responsible for pain perception - being activated by opioid peptides such as enkephalins, endorphins and dynorphins and by opiates, such as morphine. Enkephalins are naturally occurring endogenous pentapeptides with the amino acid sequence Tyr-Gly-Gly-Phe-Leu/Met. Both enkephalins are potent agonists of the delta receptor, and to a lesser extent the mu receptor, with little to no effect on the kappa receptor. Like most small peptides, enkephalins are easily catabolised via enzymatic degradation and show poor blood-brain barrier penetration. The attachment of sugars to peptides increases their penetration of the blood-brain barrier but also may affect interactions with receptors. In this study, the [Leu5]enkephalin and [Leu5]enkephalin containing the ß-D-glucuronic acid were investigated to explain how the presence of sugar moiety in the peptide molecule influences its interaction with the opioid receptors. In conclusion, the conjugation of an enkephalin molecule with the glucuronic acid has a direct and strong impact on the receptor-ligand interactions. The enhancement of ligand binding is much stronger in the delta receptor than in the mu receptor; thus, enkephalin conjugated with glucuronic acid shows greater selectivity toward the delta opioid receptor than the original peptide.