Leu-enkephalin Binding Research Articles

Computational Insights into the Interaction between Neprilysin and α-Bisabolol: Proteolytic Activity against Beta-Amyloid Aggregates in Alzheimer’s Disease

(1) Background: Alzheimer’s disease (AD) is an irreversible disorder of the central nervous system associated with beta-amyloid protein (Aβ) deposition and accumulation. Current treatments can only act on symptoms and not the etiologic agent. Neprilysin and α-bisabolol have been shown to reduce the aggregation of Aβ, suggesting a potential interaction between both molecules, leading to increased proteolytic activity on Aβ aggregates. (2) Methods: Computational simulations were conducted to explore the interaction between murine neprilysin [NEP(m)] and α-bisabolol and their effects on enzymatic activity. NEP(m) structure was predicted using comparative modeling, and the binding pattern to α-bisabolol and its effects on leu-enkephalin binding were explored through docking calculations and molecular dynamics simulations, respectively. (3) Results: The findings suggest that α-bisabolol stabilizes the Val481-Pro488 segment of NEP2(m), which directly interacts with the peptide substrate, enabling an optimized alignment between the catalytic residue Glu525 and leu-enkephalin. (4) Conclusions: This computational evidence strongly supports the notion that α-bisabolol stabilizes peptide substrates at the NEP2(m) catalytic site, leading to the positive modulation of enzymatic activity.

Structural and biological implications of the binding of Leu-enkephalin and its metal derivatives to opioid receptors

Binding of Leu-enkephalin and [Rh(III)(η(5)-Cp*)(η(6)-Tyr(1))]Leu-enkephalin to the recently published crystal structures of the μ- and δ-opioid receptor is studied. Docking of free Leu-enkephalin reveals two preferred conformations, one of which suggests an alternative binding site for the tyrosine residue. Furthermore, the three-dimensional solution structure of [Rh(III)(η(5)-Cp*)(η(6)-Tyr(1))]Leu-enkephalin was solved by using 2D NMR spectroscopic techniques.

Brain methionine- and leucine-enkephalin receptors in patients with dementia

Brain [ 3H]Met- and [ 3H]Leu-enkephalin binding was studied in patients with Alzheimer's disease (AD) and vascular dementia (VD), and in age-matched controls. Brain areas investigated were the internal and external globus pallidus, amygdala, hippocampus and temporal cortex. In AD, the binding of both enkephalins decreased in all brain areas examined, except in the external globus pallidus for both enkephalins and in the internal globus pallidus for leucine-enkephalin. Scatchard analysis of amygdaloid samples showed a decrease in the number of receptors (Bmax) without any change in their affinity ( K d). In patients with VD, no significant changes in enkephalin binding were seen. Thus, in AD, enkephalin binding (mainly reflecting delta opioid receptor subtype) is decreased, especially in limbic areas.

NMR investigations of Mn(II)-leucine-enkephalin complexes in [ 2H 6]-DMSO solution

Structural and kinetic features of the Mn(II)-Leu-enkephalin binding equilibria were delineated by measuring 13C and 1H NMR spin-lattice relaxation rates. The temperature dependence of such rates showed that some carbons were experiencing slow exchange regimes such that kinetic parameters at room temperature could be calculated (k off= 1400 sec −1, ΔH* = 12.0 kcal/mol, ΔS* = −9.9 e.u.). The paramagnetic rates of fast exchanging carbons were interpreted by the Solomon-Bloembergen-Morgan theory to provide structural parameters. The terminal carboxyl and amino groups were shown to be the binding sites. The motional correlation time(τ c = 0.6 nsec at 298 K) was calculated by measuring selective and double-selective 1H spin-lattice relaxation rates for the free peptide. The number of coordinated ligands was evaluated by considering the distance of the Leu CO in the complex at 2.54 Å, as shown by molecular models. Finally, carbon-Mn(II) distances were calculated and the molecular model of the 1:1 complex was built.

Protein-peptide interactions as probed by tryptophan fluorescence: serum albumins and enkephalin metabolites.

Binding of Leu-enkephalin and the enkephalin metabolite, tyrosine-glycine-glycine (TGG), to bovine serum albumin (BSA) was studied as a model to investigate protein peptide interactions. TGG and Leu-enkephalin quench the tryptophyl fluorescence of BSA. Stern-Volmer quenching constants were typically in the range of 40 to 300 M-1, depending on the experimental conditions. The addition of Cu(II) or Ni(II) did not change the quenching constant, indicating that TGG does not compete for the metal binding sites on BSA. From fluorescence quenching studies with TGG, tyrosyl-glycine, tyrosine and glycyl-glycine, it was concluded that the presence of the tyrosine residue is required for the observed quenching. The phenolic group in tyrosine accounted for the quenching mechanism because phenol was efficient in quenching BSA fluorescence, whereas phenylalanine had no detectable effect. A large solvent isotope effect on the quenching constant of phenol and TGG with BSA strongly suggests an active role of the -OH functionality in the quenching mechanism.

An approach to vectorisation of pharmacologically active molecules: The covalent binding of leu-enkephalin to a modified β- cyclodextrin.

The neurotropic peptide Leu-enkephalin has been coupled to a mono-6-amino permethyl βcyclodextrin at the C-terminal residue. The resulting compound has been fully characterized by proton NMR in D 2O and d6-DMSO evidencing complete reduction of the molecular symmetry of the cyclodextrin.

Transport of leucine-enkephalin across the blood-brain barrier in the perfused guinea pig brain.

Transport of [tyrosyl-3,5-3H]enkephalin-(5-L-leucine) [( 3H]Leu-enkephalin) across the blood-brain barrier was studied in the adult guinea pig, by means of vascular perfusion of the head in vivo. The unidirectional transfer constant (Kin) estimated from the multiple-time uptake data for [3H]Leu-enkephalin ranged from 3.62 X 10(-3) to 3.63 X 10(-3) ml min-1 g-1 in the parietal cortex, caudate nucleus, and hippocampus. Transport of [3H]Leu-enkephalin was not inhibited by unlabelled L-tyrosine (the N-terminal amino acid) at a concentration as high as 5 mM, or by the inhibitor of aminopeptidase activity bacitracin (2 mM), suggesting that there was no enzymatic degradation of peptide at the blood-brain barrier. By contrast, 2 mM unlabelled Leu-enkephalin strongly inhibited the unidirectional blood-to-brain transport of [3H]Leu-enkephalin by 74-78% in the parietal cortex, caudate nucleus, and hippocampus. The tetrapeptide tyrosyl-glycyl-glycyl-phenylalanine (without the C-terminal leucine of Leu-enkephalin), at a concentration of 5 mM, caused a moderate inhibition ranging from 15 to 29% in the brain regions studied, whereas the tetrapeptide glycyl-glycyl-phenylalanyl-leucine (without the N-terminal tyrosine) at 5 mM was without effect on Leu-enkephalin transport. Unidirectional brain uptake of Leu-enkephalin was not altered in the presence of naloxone at a concentration as high as 3 mM (1 mg/ml), suggesting that there is no binding of Leu-enkephalin to opioid receptors at the blood-brain barrier. It is concluded that there is a specific transport mechanism for Leu-enkephalin at the blood-brain barrier in the guinea pig.(ABSTRACT TRUNCATED AT 250 WORDS)

Specific binding of leu-enkephalin to small and large intestinal epithelial cells from guinea-pig

1. 1. Leu-enkephalin receptors were identified in guinea-pig intestinal mucosa in small as well as in large epithelial cells. 2. 2. Binding studies at apparent equilibrium could be interpreted in terms of two populations of receptors in every intestinal segment. 3. 3. Leu-enkephalin receptors were unequally distributed along the intestinal mucosa, with the lowest density but the highest affinity values in the caecum and colon. 4. 4. Duodenal epithelial cells exhibited the highest binding values due to a great number of low-affinity receptors. 5. 5. Receptors exhibited a high degree of specificity for Leu-enkephalin as evidenced by the poor competition shown by a variety of enkephalin analogues and naloxone and the lack of effect of other unrelated peptides present in the intestinal tract.

Interaction of Leu-enkephalin with isolated enterocytes from guinea pig: Binding to specific receptors and stimulation of cAMP accumulation

The specific binding of Leu-enkephalin and the stimulatory effect of the peptide on cAMP accumulation have been assessed in isolated enterocytes of guinea pig. The binding was reversible as well as time and temperature dependent. Two classes of binding sites could be defined: a class with a relatively high affinity (K d=0.7 μM) that represented 1% of total binding capacity, and another class with low affinity (K d=55.5 μM). The stimulation of cAMP accumulation was also shown to depend on time and temperature and was potentiated by a phosphodiesterase inhibitor. Half-maximal stimulation of cAMP accumulation was observed at 119 μM and maximal stimulation (27-fold basal level) at 300 μM Leu-enkephalin. Both steps of the interaction were not modified by Na + but exhibited a high specificity since modifications in the structure of Leu-enkephalin resulted in an important loss of binding affinity and stimulatory activity.

Leu-enkephalin binding to cultured human T lymphocytes

Evidence is given for the existence of leu-enkephalin binding activity on the cell surface of a human lymphoid T line, our data suggest that binding is due to receptor structures which in all likelyhood are lipoproteic, as is already known to be the case with the receptors present in the mammalian brain. In spite of these similarities, however, lymphocytes exhibit anomalous specificity as far as the binding toward opioid alkaloids and their inhibitors is concerned.

Leu-enkephalin binding to cultured human T lymphocytes

Evidence is given for the existence of leu-enkephalin binding activity on the cell surface of a human lymphoid T line. Our data suggest that binding is due to receptor structures which in all likelyhood are lipoproteic, as is already known to be the case with the receptors present in the mammalian brain. In spite of these similarities, however, lymphocytes exhibit anomalous specificity as far as the binding toward opioid alkaloids and their inhibitors is concerned.

3H]Leu-enkephalin binding following chronic swim-stress in mice

Warm water swimming produces in mice an opiate-like antinociceptive response. Chronic swimming produces tolerance to the antinociceptive response and, depending on the schedule, cross-tolerance with morphine and naloxone intensified withdrawal signs. Low affinity [ 3H]Leu-enkephalin binding to brain homogenates at low temperature was significantly reduced in acutely swum mice and chronically swum mice whether or not they were swum. Preincubation at 37°C abolished all between-group differences. Results following chronic swimming were similar whether or not the schedule produced morphine cross-tolerance. These results were discussed in terms of the interpretation that reduced binding reflects increased in vivo occupation of opioid binding sites.

Regional specificity of changes in [ 3H]Leu-enkephalin binding associated with warm water swimming in mice

Warm water swimming produces in mice an opiate-like antinociceptive response. Immediately following a 3 min swim, mice were sacrificed by immersion in liquid nitrogen and three brain areas dissected from frozen tissue: basal ganglia (#1), periventricular, thalamic and ventral areas (#2), and medulla-brainstem (#3). A 37% reduction in exogenous [ 3H]Leu-enkephalin binding was observed in section #2. When section #2 was further divided, a 41% reduction was observed in an area containing opiate receptor contributions from interpeduncular nucleus, thalamus, hypothalamus and amygdala. These results are consistent with the association of opiate receptors in these regions with swim-induced antinociception.

Evolution of striatal opiate receptors

The phylogenetic trend in the relative proportions of two opiate receptor subtypes from invertebrates to humans was identified. The binding of tritiated leu-enkephalin and dihydromorphine was compared in cryostat-cut striatal sections from 11 vertebrates and the anterior ganglion from 2 arthropods. As an independent measure of species relatedness, we used the deviations from the human sequence of amino acids in the molecule cytochrome-C. We show that the percentage of leu-enkephalin binding decreases as the relatedness to human increases.

β‐Endorphin

Human beta-endorphin analogs with various chain lengths have been investigated for their potency in displacing tritiated dihydromorphine and Leu-enkephalin binding in rat brain membrane preparations. It was found that the reduction of chain length from residues 1-31 to 1-5 led to a gradual loss of preference for the morphined receptor. In addition, the extension of the chain length of the Met-ekephalin segment to the COOH-terminal glutamic acid modified the binding of the NH2-terminal sequence to the enkephalin receptor. The fact that camel beta-endorphin is more potent in displacing the two tritiated primary ligands than the human hormone is also reported herein.

Inhibition of leu-enkephalin binding by neuroleptics.

The binding of leu-enkephalin to synaptosome-enriched fractions prepared from rat whole brain has been measured in the presence and absence of haloperidol, chlorpromazine and chlozapine. All three neuroleptics produced a dose-dependent inhibition of the binding with inhibition constants of 33.7, 89.6, and 34.2 microM for haloperidol, chlorpromazine, and clozapine, respectively, and resulted in parabolic Hill plots suggestive of multiprocess modes of action rather than simple competitive or noncompetitive interactions. Except for the case of haloperidol, the inhibition constants are comparable to brain tissue levels of neuroleptics after chronic administration. This suggests that neuroleptic treatment may block a significant fraction of endogenous leu-enkephalin from reaching its CNS receptors.

The correlation between swim-stress induced antinociception and [ 3H] leu-enkephalin binding to brain homogenates in mice

Mice which had been made to swim for 3 minutes showed a tail flick latency which was significantly longer than that of unswum controls. The [ 3H] leu-enkephalin [LE] binding to brain homogenates from swum mice was significantly reduced when compared with that from unswum controls. Scatchard analysis revealed that the reduction in binding occurred at the LE low affinity site. However, when homogenates were allowed a preincubation period of 20 min at 37°C, the difference in LE binding between swum and unswum mice was no longer apparent. These data are interpreted to suggest that the reduced LE binding may be due to the occupation of a proportion of the opiate receptor population by an endogenous ligand. A correlation between the duration of the swim induced antinociceptive response and the changes in LE binding is described which although non-significant, is consistent with the interpretation for the involvement of endogenous opiates in the observed increases in tail flick latency.

Pharmacological evidence of a possible tryptaminergic regulation of opiate receptors by using indalpine, a selective 5-HT uptake inhibitor

Indalpine or LM 5008 (4-[2-(3-indolyl)ethyl]piperidine), a selective and potent inhibitor of the uptake of 5-hydroxytryptamine, showed analgesic properties in the hot plate test in mice. It enhanced the analgesic effect of morphine and pethidine. This activity could not be explained by alterations of the pharmacokinetics of morphine since the brain morphine level was not changed by pretreatment with indalpine. This drug also antagonized the development of tolerance to morphine in mice. Morphine was 3–7 times more potent in competing for [ 3H]-naloxone (−NaCl), [ 3H]-metenkephalin or [ 3H]-leu-enkephalin binding when animals were pretreated in vivo with indalpine although indalpine itself had no effect on opiate receptor binding sites. In vitro addition of indalpine or 5-HT did not change the activity of morphine on opiate receptor binding sites. In vivo administration of indalpine sensitized the agonist state of opiate receptor binding sites, as measured by [ 3H]-naloxone (−NaCl) for example, to enkephalin without changing the antagonist state, as measured by [ 3H]-naloxone (+NaCl). The effect of indalpine was antagonized by the 5-HT synthesis inhibitor PCPA. Moreover, indalpine antagonized the depletion of opioid peptides induced by cyclophosphamide. All these results could suggest a tryptaminergic regulation of opiate receptors.

Effect of autoanalgesia on CNS enkephalin receptors

Fear conditioning to foot shock (15 sec/day, 12 days) elicited autoanalgesia in 10 male (Sprague Dawley) rats, while 17 non-shock control rats exhibited no analgesia as measured by the tail-flick assay. The binding of 3H-leu-enkephalin to synaptosomal preparations isolated from fear conditioned (experimental) and control animals was analyzed. At leu-enkephalin concentrations of 10 −9 M or less, both synaptosomal preparations demonstrated high affinity binding with dissociation constants on the order of 10 −10. Binding of leu-enkephalin could not be displaced by a hundred-fold excess of naloxone at leu-enkephalin concentrations less than 10 −9 M. However, the ability of naloxone to compete with leu-enkephalin for binding sites progressively increased at concentrations greater than 10 −9 M leu-enkephalin. At these ligand concentrations, the competition of naloxone for leu-enkephalin binding sites was more dramatic in the control than in the experimental animals. These data support the existence of two classes of receptors for leu-enkephalin, one of which is not blocked by opiate antagonists. Furthermore, changes in binding capacity associated with autoanalgesia produced by conditioned fear are consistent with the hypothesis of endogenous release of opiate-like peptides in response to stress.