Degradation Of Met-enkephalin Research Articles

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.

1-Substituted sialorphin analogues\u2014synthesis, molecular modelling and in vitro effect on enkephalins degradation by NEP

Rat sialorphin (Gln-His-Asn-Pro-Arg) is a natural blocker of neprilysin (NEP) that belongs to the family of endogenous opioid peptide-degrading enzymes. Studies have confirmed the efficiency of sialorphin in blocking the activity of NEP, both in vitro and in vivo. It has been demonstrated that this inhibitor has a strong analgesic, anti-inflammatory, immunological and metabolic effect either directly or indirectly by affecting the level of Met/Leu-enkephalins. In this work, sialorphin and their 12 analogues were synthesised using the solid-phase method. The effect of the peptides on the degradation of Met-enkephalin by NEP and metabolic degradation in human plasma was investigated in vitro. We show that the change in the N-terminal amino acid configuration from l to d in almost all peptides, except d-Arg-His-Asn-Pro-Arg (peptide XI), led to the abolition of their inhibitory activity. With molecular modelling technique we explained the structural properties of the l and d-arginine located on the N-terminal part of the peptide. The detailed analysis of the protein binding pocket allowed us to explain why d-arginine is so unique among all d residues. Peptide XI showed the highest stability among the tested peptides in human plasma. For instance sialorphin after a 2-hour incubation in human plasma was almost completely decomposed, while the level of peptide XI dropped to 45% after 48 h under these conditions.

Alanine scan of sialorphin and its hybrids with opiorphin: synthesis, molecular modelling and effect on enkephalins degradation

Enkephalins are involved in a number of physiological processes. However, these peptides are quickly degraded by peptidases, e.g. the neutral endopeptidase (NEP). Inhibition of the enzymatic degradation of enkephalins is one of the possible approaches to prolong their activity. Selective inhibitor of NEP, sialorphin, is the attractive lead compound for enkephalins degradation studies. In this work, an alanine scan of sialorphin and a series of its hybrids with opiorphin, synthesised by the solid phase method, were performed. The effect of the peptides on degradation of Met-enkephalin by NEP in vitro was investigated. Molecular modelling technique was used to identify residues responsible for protein–ligand interactions. We showed that substitution of amino acids Gln1, Pro4 and Arg5 of sialorphin for Ala significantly reduced the half-life of Met-enkephalin in the presence of NEP. [Ala3]sialorphin displayed a higher inhibitory potency against NEP than sialorphin. Substitution of His2 for Ala led to a compound which was as active as lead compound. Sialorphin has a structure which hardly tolerates substitution in its sequence at positions 1, 4 and 5. The conversion of His2 for alanine in sialorphin is tolerated very well. The higher inhibitory potency of [Ala3]sialorphin than sialorphin against NEP is caused by removal of the hydrophilic residue (Asn) and a better fit of the peptide to the enzyme-binding pocket. The role of side chains of sialorphin in degradation of enkephalin by NEP has been explored. This study also provides an important SAR information essential for further drug design.

Methionine enkephalin suppresses metabolic activity of a leukemic cell line (NALM-1) and enhances CD10 expression

NALM-1 cells (a cell line derived from human pre-B leukemia) were exposed to the opioid pentapeptide methionine–enkephalin (Met–enkephalin) and/or to thiorphan, an inhibitor of the enzyme that degrades the enkephalins (membrane endopeptidase EC 3.4.24.11, CALLA, the CD10 marker). Metabolic and proliferative activity was assessed after 6, 24 and 48 h in microplates using a colorimetric assay with vital dye MTT. CD10 expression was determined by means of semi-quantitative RT-PCR. Exposure to the Met–enkephalin at concentrations of 10 −8–10 −6 M for 6 h reduced the MTT-activity, and after 24 and 48 h the suppression waned. Thiorphan (5×10 −6 M) abrogated the suppressive effect of the enkephalin, and after 6 h converted suppression into stimulation. Met–enkephalin (10 −6 M) increased and thiorphan (2.5×10 −6–10 −6 M) decreased expression of CD10 at the RNA level. Suppression of the MTT uptake was attributed to the products of Met–enkephalin degradation caused by the enzymatic activity of CD10.

Regional metabolism of Met-enkephalin and cholecystokinin on intact ratbrain slices: characterization of specific peptidases.

The metabolism of Met-enkephalin and cholecystokinin (CCK) 8-(sulfated) by intact microslices was studied in rat brain regions. Incubation of brain slices with Met-enkephalin (400 microM) resulted in a linear rate of disappearance of parent peptide and appearance of metabolic fragments whose rate of accumulation was specific to brain region. The degradative rate (pmol/min/mg of protein) of Met-enkephalin was high in caudate-putamen (5,160 +/- 120) and lower in nucleus accumbens (3,630 +/- 110) and frontal cortex (3,180 +/- 120). Inhibition of aminopeptidases decreased Met-enkephalin degradation (50-97% vs. control) in frontal cortex but was less effective in caudate-putamen (20-34%). Tyr-Gly-Gly and Phe-Met were recovered in caudate-putamen and nucleus accumbens, whereas negligible quantities of these fragments were recovered from frontal cortex. Phosphoramidon, an inhibitor of neutral endopeptidase 24.11, decreased Met-enkephalin degradation in caudate-putamen (14%) but had no effect on that in frontal cortex. A cocktail of bestatin or leuhistin (inhibitors of aminopeptidases), phosphoramidon, and captopril (an inhibitor of angiotensin converting enzyme) protected Met-enkephalin from degradation (recovery > 95%) in caudate-putamen. CCK 8-(sulfated) degradation on slices from caudate-putamen, nucleus accumbens, and frontal cortex was not altered by inhibitors of neutral endopeptidase 24.11, metalloendopeptidase 24.15, angiotensin converting enzyme, or thiol proteases. Inhibitors of either aminopeptidases or serine proteases produced small reductions (13-30%) in CCK degradation in each region. These data provide evidence for regional and structural specificity in terminating the actions of neuropeptides.

Effect of X-Prolyl Dipeptidyl Aminopeptidase Deficiency on Lactococcus lactis

The genetic determinant (pepXP) of an X-prolyl dipeptidyl aminopeptidase (PepXP) has recently been cloned and sequenced from both Lactococcus lactis subsp. cremoris (B. Mayo, J. Kok, K. Venema, W. Bockelmann, M. Teuber, H. Reinke, and G. Venema, Appl. Environ. Microbiol. 57:38-44, 1991) and L. lactis subsp. lactis (M. Nardi, M.-C. Chopin, A. Chopin, M.-M. Cals, and J.-C. Gripon, Appl. Environ. Microbiol. 57:45-50, 1991). To examine the possible role of the enzyme in the breakdown of caseins required for lactococci to grow in milk, integration vectors have been constructed and used to specifically inactivate the pepXP gene. After inactivation of the gene in L. lactis subsp. lactis MG1363, which is Lac and Prt, the Lac Prt determinants were transferred by conjugation by using L. lactis subsp. lactis 712 as the donor. Since growth of the transconjugants relative to the PepXP strains was not retarded in milk, it was concluded that PepXP is not essential for growth in that medium. It was also demonstrated that the open reading frame ORF1, upstream of pepXP, was not required for PepXP activity in L. lactis. A marked difference between metenkephalin degradation patterns was observed after incubation of this pentapeptide with cell extracts obtained from wild-type lactococci and pepXP mutants. Therefore, altered expression of the pepXP-encoded general dipeptidyl aminopeptidase activity may change the peptide composition of fermented milk products.

Degradation of Met-enkephalin by hemolymph peptidases in Mytilus edulis.

1. Met-enkephalin is degraded by peptidases present in the hemolymph fluid and hemocyte membrane suspension of Mytilus edulis. Degradation of Met-enkephalin is rapid in the fluid and slower in the membrane. 2. Aminopeptidase activity is bestatin sensitive in hemocyte membrane and highest in the fluid of the hemolymph, which appears to have a component which is insensitive to inhibitor. 3. ACE activity is found only in the fluid of the hemolymph. 4. Carboxypeptidase and NEP (CD10: "enkephalinase") are membrane bound and the former appears to predominate. Phosphoramidon inhibits not only NEP, as expected, but the invertebrate carboxypeptidase as well.

Prolonged antinociceptive activity of pseudodipeptide analogues of Lys-Trp(Nps) and Trp(Nps)-Lys

Peptide bond susbstitution in the molecules of Lys-Trp(Nps) (LTN) and Trp(Nps)-Lys (TNL) by an aminomethylene and ketomethylene bond, respectively, afforded pseudodipeptides with analgesic activity. The new compounds Lysψ(CH 2NH)-Trp(Nps)-OMe (LTNAM) and Trp(Nps)ψ(COCH 2)(R,S)-Lys (TNLKM) induced a dose-dependent and naloxone-reversible analgesia following intracerebroventricular (ICV) administration to mice. The antinociceptive effects were longer lasting compared to those induced by the parent compounds. The pseudodipeptides protected Met-enkephalin degradation by rat striatal slices and, combined with an ineffective dose of the opioid peptide, induced analgesia. LTNAM and TNLKM were as potent as LTN to inhibit brain aminopeptidase in vitro and ex vivo. An increased resistance to proteolysis of the pseudodipeptides may explain their prolonged analgesic activity.

Intrathecal administration of thiorphan and bestatin enhances the antinociception and release of Met-enkephalin induced by β-endorphin intraventricularly in anesthetized rats

Effects of bestatin and thiorphan administered intrathecally, on inhibition of the tail-flick response and the release of Met-enkephalin induced by β-endorphin administered intraventricularly were studied in anesthetized rats. Intrathecal pretreatment with 100 μg of thiorphan or bestatin potentiated the inhibition of the tail-flick response induced by β-endorphin injected intraventricularly in pentobarbital anesthetized rats; the ED50 values for β-endorphin were decreased 5- and 7-fold by thiorphan and bestatin, respectively. To determine if the potentiating effect was due to the inhibition of the degradation of Met-enkephalin released by intraventricular β-endorphin, the effects of intrathecal perfusion with thiorphan or bestatin on the release of immunoreactive Met-enkephalin from the spinal cord by intraventricular injection of β-endorphin were studied. β-Endorphin injected into the 4th ventricle at a dose of 5 μg increased immunoreactive Met-enkephalin in the spinal perfusate in urethane-anesthetized rats. Thiorphan or bestatin (1 × 10 −7 to 1 × 10 −4M each) increased the amount of immunoreactive Met-enkephalin released by intraventricular β-endorphin in a dose-dependent manner. The results provede additional evidence for the hypothesis that antinociception induced by β-endorphin is mediated by release of Met-enkephalin.

Major rat brain membrane-associated and cytosolic enkephalin-degrading aminopeptidases: comparison studies.

The major cytosolic and membrane-associated enkephalin-degrading aminopeptidases were purified in parallel by column chromatography successively on DEAE-cellulose, AH-Sepharose, hydroxylapatite, Sephadex G-200, Affigel Blue, AH-Sepharose, and hydroxylapatite. With the final hydroxylapatite column, the cytosol (S) and the membrane (M) enzymes could each be resolved into two peaks, one eluted with 0.05 M phosphate (SI, MI) and the other with 0.25 M phosphate (SII, MII). The overall purification, with Arg BNA as substrate, for the SI and MI was about 450-fold; for SII and MII, 1,200-fold. The yield for each enzyme was about 2%. the major protein integral units of the four enzymes are similar; they are single polypeptide chains with a molecular weight of 100,000 daltons. Their pH optimum, substrate specificity, and sensitivity to puromycin show that they are similar to lysosomal aminopeptidase (EC 3.4.11.-). The two forms of the cytosol and the membrane enzymes have slightly different kinetic constants. With the inhibitors, SII is more sensitive to proctolin, whereas MII is more sensitive to bestatin and Arg-Phe-Ala. Mn2+ activates SI on Met-enkephalin degradation, but inhibits SII, MI, and MII.

Pharmacology of enkephalinase inhibitors: animal and human studies.

We have shown that a number of compounds which inhibit the degradation of met-enkephalin can produce naloxone-reversible analgesia in mice. These compounds also potentiate the analgesia produced by acupuncture, foot shock, and transcutaneous nerve stimulation in animals and humans. The potency of their effectiveness as analgesics or potentiators parallels their potency as inhibitors of mouse brain enkephalinase. D-Phenylalanine (DPA), one of these enkephalinase inhibitors, has been used successfully for the management of chronic intractable pain in humans and to potentiate the treatment of many painful conditions by acupuncture. Other aspects of pharmacology of DPA will be discussed, including its effects on the cardio-vascular system, behavior, and lack of development of tolerance and dependence when used chronically in animals and humans.

Processing and degradation of Met-enkephalin by peptidases associated with rat brain cortical synaptosomes.

More than 20 peptides have been identified in neurones of the brain, spinal cord and in the periphery. There is growing evidence that some of these peptides act as neurotransmitters in the nervous system (for review see Hökfelt et al., 1980, Snyder, 1980, Konishi et al., 1981). The discovery of the enkephalins, Tyr-Gly-Gly-Phe-Met and Tyr-Gly-Gly-Phe-Leu, (Hughes et al., 1975) has led to numerous studies of their metabolism. For the regulation of the activity of these peptides the mode of processing from larger precursor proteins of peptides and of inactivation by proteolytic cleavage is of fundamental importance. Enkephalin containing polypeptides have been described in different organs such as adrenal medulla, intestine mucosa and brain (Lewis et al., 1979, Stern et al., 1981) as potential processing products of the larger pro-enkephalin precursor. The carboxyterminus of pro-enkephalin consists of the met-enkephalin-Arg6-Phe7 sequence (Gubler et al., 1982, Noda et al., 1982).KeywordsAdrenal MedullaSynaptosomal FractionPeptide HydrolaseSynaptosomal ProteinDipeptidyl CarboxypeptidaseThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Processing and degradation of met-enkephalin by peptidases associated with rat brain cortical synaptosomes

The generation of met-enkephalin (Tyr 1-Gly 2-Gly 3-Phe 4-Met 5) from met-enkephalin-Arg 6-Phe 7 and subsequent degradation of the liberated peptides to the free amino acids by rat brain cortical synaptosomes in vitro was demonstrated by HPLC and amino acid analyses. Kinetic measurements of the individual steps of met-enkephalin processing and degradation upon incubation with synaptosomes revealed the following sequence of cleavage: 1. Hydrolysis of the Met 5-Arg 6 peptide bond, generating met-enkephalin and the dipeptide Arg-Phe. Captopril and EDTA inhibit this reaction. 2. Hydrolysis of the Tyr 1-Gly 2 peptide bond, generating Tyr and a tetrapeptide. Puromycin (ID 50 = 5 × 10 −5 m) and parahydroxymercuribenzoate (ID 50 = 5 × 10 −4 m) inhibit this reaction. 3. Hydrolysis of the Gly 3-Phe 4 peptide bond. Parahydroxymercuribenzoate (ID 50 = 5 × 10 −4 m) inhibits this reaction completely. 1 mmol liter −1 Puromycin does not inhibit this reaction. 4. Hydrolysis of the Phe 4-Met 5 peptide bond. 5. Hydrolysis of the Gly 2-Gly 3 peptide bond. The pH optimum of all cleavage reactions was found to be around 7.8.

Met-enkephalin binding to opiate receptors is not functionally coupled to biodegradation

Using synaptosomal rat brain membranes, the degradation of Met-enkephalin in both free and receptor-bound form was measured, together with the dissociation of Met-enkephalin from the receptors. The results show that the degradation rate of initially receptor-bound Met-enkephalin is significantly smaller than either the rate of dissociation from the receptors or that of the degradation of free Met-enkephalin. These data suggest that intact Met-enkephalin dissociates from the receptors and then it becomes a target for the same membrane-associated peptidases that split free Met-enkephalin. This view is supported by the good fitness of the measured degradation of initially receptor-bound Met-enkephalin to the predicted degradation curve calculated from the dissociation rate of receptor-bound Met-enkephalin and the degradation rate of free Met-enkephalin.

Enkephalin-hydrolyzing peptidases of rat brain membranes: Are they topographically/functionally coupled to opiate receptors?

At least four peptidases are involved in the breakdown of Met-enkephalin by rat brain membranes: aminopeptidase(s), angiotensin-converting enzyme (ACE), metalloendopeptidase, and dipeptidyl aminopeptidase. Opiates and some metabolically stable enkephalin analogs used at high concentrations to displace Met-enkephalin from the receptors have no significant effect on the degradation of Met-enkephalin by these peptidases. There is no difference between the dissociation rates of Met-enkephalin from the opiate receptors in membranes, with full and with completely blocked aminopeptidase, ACE, and metalloendopeptidase activities. These data do not imply a close topographical coupling between the opiate receptors and the peptidases studied.

Neuropeptide degrading enzyme(s) in plasma and brain: Effects of in vivo neuroleptic administration

Summary Degradation of Met-enkephalin (Try-Gly-Gly-Phe-Met) by clevage of the Tyr-Gly bond is enhanced in rodent plasma during a single or multiple dose regimen of chlorpromazine (CPZ). This is observed after five hours from time of last dose and persists as long as twenty hours later. Enhanced plasma protease activity was also observed with fluphenazine but not with imipramine. In contrast, levels of membrane-bound aminopeptidase activity in corpus striatum did not differ significantly after three weeks treatment with CPZ. In Vitro additions of CPZ (up to 100μM) to the plasma and brain preparations did not alter enzyme activity.

β-Endorphin inhibits met-enkephalin breakdown by a brain aminopeptidase: Structure-activity relationships

An aminopeptidase solubilized and isolated from rat brain membranes selectively splits the Tyr 1-Gly 2 peptide bond of Met-enkephalin. β h-Endorphin, which is apparently resistant to the aminopeptidase, inhibited the action of this peptidase on Met-enkephalin degradation competitively; the K i value was 11.5 μM. Arg 0-β h-endorphin was found to be 10 times more potent than β h-endorphin. From further structure-activity data it is concluded that the N-terminal amino group and some residues within region 18–31 of the β-endorphin structure are cooperatively involved in binding to the active site of the aminopeptidase.

Degradation of Met-enkephalin by extracts of various regions of the human brain: Effects of antipsychotics and narcotics in vitro

Antischizophrenic drugs, reduced in a concentration-dependent fashion enkephalin degradation by the soluble and particulate fractions of the human cerebral cortex and cerebellum. The order of potency is as follows: thioridazine > chlorpromazine > fluphenazine > haloperidol ≧ promazine with IC 50 of 50, 80, 120, 200–250 μM, respectively. Kinetic studies revealed non-competitive and competitive inhibition by thioridazine and chlorpromazine, respectively. Narcotics, were weak inhibitors of enkephalin degradation. For dl-, d-, l-methadone and l-α-acetylmethadol, IC 50 was about 500 μM, and 1000 μM for heroin and morphine. It is suggested that inhibition of the degradation of endogenous morphinomimetic peptides in the CNS may be a crucial factor governing the pharmacology of some neuroleptics and other psychoactive drugs. Enkephalin-hydrolyzing activity was ubiquitous and exhibited considerable regional differences in the normal human and in Huntington's chorea brains. The rate of enkephalin degradation is generally higher in the subcortical nuclei than in the cortex and cerebellum. The highest hydrolytic activity was found in the substantia nigra, anterior thalamus, septal area, globus pallidus and caudate nucleus, in this decreasing order.

The effects of biogenic amines on cleavage of met-enkephalin by brain tissue extracts

Abstract 1. The biogenic amines vary largely in their ability to inhibit the degradation of metenkephalin by the soluble fraction of human brain cortex and by the high and low affinity particulate bound peptidase of rat striatum. 2. The hydrolyzing activity of the cortical soluble fraction was inhibited by amines in this order of decreasing potency: DA, 6-OHDA, L-NA, tryptamine, 5-HT and 5-methoxy-tryptamine with IC 50 of 40, 40, 200, 250, 400 and 450 μM, respectively. 3. With high affinity peptidase of rat striatum, the order of inhibitory activity was as follows: DA, L-NA, DL-NA, 3-0-methyldopamine, octopamine and 5-HT, with IC 50 of 20, 60, 100, 100, 250 and 260 μM, respectively. 4. Biogenic amines may have an essential and direct regulatory role in the metabolism of enkephalin in the CNS.

INHIBITION OF THE ENZYMATIC DEGRADATION OF MET-ENKEPHALIN BY CATECHOLAMINES

The influence of the catecholamine hormones and some of their metabolites on the enzymatic degradation of met-enkephalin by rat brain homogenates has been evaluated. The enzyme activity was determined by the release of [125I]-tyrosine from radioiodinated met-enkephalin following Porapak Q column chromatography. Each of the biologically active catecholamines inhibited the measured enzyme activity, and dose-response curves indicated that dopamine was most effective and epinephrine least effective. At 5 x 10(-5)M the O-methylated metabolite of each hormone failed to inhibit the enzyme activity. An enzyme activity similar to that found in brain was also found in adrenal and ileal homogenates. This enzyme activity was strongly inhibited by dopamine as well as by puromycin.